Oligosaccharide compound for inhibiting intrinsic coagulation factor x-enzyme complex, and preparation method therefor and uses thereof

ABSTRACT

A purified oligosaccharide compound having antithrombotic activity or a mixture of a homologous compound thereof and a pharmaceutically acceptable salt thereof, a preparation method for the mixture, a pharmaceutical composition containing the mixture, and uses thereof serving as an intrinsic factor X-enzyme (Xase) inhibitor in the preparation of drugs for preventing and/or treating thrombotic diseases.

FIELD OF THE INVENTION

The present invention belongs to the technical field of medicine, and inparticular relates to a purified oligosaccharide compound havingantithrombotic activity or a mixture of homologous compounds thereof,and a pharmaceutically acceptable salt thereof, a preparation method anduses thereof.

BACKGROUND OF THE INVENTION

Thromboembolic diseases including ischemic stroke, coronary heartdisease, venous thromboembolism are the major lethal causes of humanbeings. Anti-thrombotic drugs such as fibrinolytic, anticoagulant andantiplatelet drugs are the basic means for clinical drug prevention andtreatment of thrombotic diseases, but existing antithrombotic drugs havecommon defects: bleeding tendency and serious bleeding risk. Reducingbleeding tendency and bleeding risk is the core goal of the developmentof new antithrombotic drugs. Researches in recent years have found thatintrinsic coagulation pathways are closely related to pathologicalthrombosis, and may not be necessary for hemostasis. Therefore,intrinsic coagulation factor inhibitors have become the focus ofresearch on antithrombotic drugs with low bleeding tendency. Among them,preclinical and clinical trial studies on coagulation factor XIIa, XIaand IXa inhibitors have been carried out in succession. The intrinsicfactor tenase complex (Xase) is the final and rate-limiting enzyme ofthe intrinsic coagulation pathway, and its selective inhibitor hasimportant potential clinical application value.

Fucosylated glycosaminoglycan (FG) is a glycosaminoglycan with uniquechemical structures and pharmacological activities found up to nowexclusively in echinoderms, which has a chondroitin sulfate-likebackbone, and sulphated fucosyl (Fuc)-substituted side chains (Yoshidaet. al, Tetrahedron Lett, 1992, 33: 4959-62; Mourão et. al, J Biol Chem,1996, 271: 23973-84). Studies have shown that native FG has potentanticoagulant activity, and its anticoagulant mechanism is mainlyrelated to inhibition of intrinsic Xase activity (Thromb Haemost, 2008,100: 420-8; J. Biol. Chem., 1996, 271: 23973-84). However, native FG hasextensive and contradictory pharmacological effects, including inductionof platelet aggregation and induced decrease in circulating plateletcount, activation of XII and such a side effect may cause hypotension,and so on (Thromb Haemost, 1988, 59: 432-4; Thromb Haemost, 2010, 103:994-1004). Thus the application value of FG under systemicadministration is limited. Properly depolymerized FG can reduce theactivity of induction of platelet aggregation (Thromb Haemost, 1991, 65:369-73), thereby increasing its selectivity for inhibition of theintrinsic factor Xase.

The present inventors have previously systematically studied thechemical depolymerization of native FG and the chemical andpharmacological properties of the depolymerized product. For example,the Chinese invention patent CN 101724086 B discloses a peroxidativedepolymerization method of FG and the obtained depolymerized product caninhibit thrombus formation and the bleeding tendency is significantlyreduced, however, the product obtained by the method is difficult to befurther isolated and purified due to the complicated terminal structure.

It has been found now that the native FG obtained by extracting from thebody wall of echinoderms by a conventional method usually also containsglucan, fucan and/or hexosamine-containing polysaccharide compounds,which have a molecular weight distribution like that of native FG Thesepolysaccharides in native FG are difficult to be removed completely bygel chromatography, ultrafiltration or even ion exchange chromatography.A comparative study conducted by the present inventors has shown thatusing the technical methods described in the above literatures, thenatural FG extracted from the echinoderms usually contains otherpolysaccharide compositions in a mass ratio of about 10%-20%. Since allof these polysaccharide compositions could be depolymerized byperoxidation, the oligosaccharide compositions of the peroxidativedepolymerization product of natural FG are quite complicated.

Chinese invention patent CN 103214591A discloses adeacetylation-deaminative depolymerization method of FGS which canselectively cleave D-acetylgalactosamine (GalNAc)-(β1→4)-D-glucuronicacid (D-GlcA) glycosidic bond, obtaining a depolymerized productcontaining 2,5-anhydro-D-talose (anTal) at the reducing terminal. Theoligosaccharide homologues in the obtained depolymerized product arecomposed of 3m monosaccharide residues (m is a natural number,hereinafter the same). The deacetylation-deaminative depolymerizationmethod cannot depolymerize glucan and fucan mixed in natural FGS andafter the deacylative-deaminative depolymerization treatment, theseundepolymerized polysaccharide impurities can be easily removed by gelchromatography or ultrafiltration method. The deacylative-deaminativedepolymerization product of natural FG has more regular structuralfeatures, and the oligosaccharide contained therein may be furtherisolated and purified (CN 104370980A). Studies have shown that among thehomologous oligosaccharide compounds obtained by deacylative-deaminativedepolymerization of natural FGS nonasaccharide (NSac) is the the minimumfragment with potent inhibitory activity against Xase (Proc Natl AcadSci USA. 2015; 112(27): 8284-9).

The present inventors' granted patent CN 201310099800 discloses aβ-eliminative depolymerization method of native FG The method comprisestreating FG carboxylate with a base in a non-aqueous solvent toselectively cleave D-GalNAc-(β1→4)-D-GlcA glycosidic bond, therebyobtaining a depolymerized product containing unsaturatedΔ^(4,5)-hexuronic acid group (ΔUA) at the non-reducing terminal, and thedepolymerized product is a mixture of a series of oligosaccharidecompounds. Similarly, since the 3-elimination depolymerization methodhas an excellentglycosidic bond selectivity, it cannot depolymerizeother types of polysaccharides contained in the native FG extract,thereby facilitating the removal of non-FG polysaccharide impuritiesfrom the FG extract.

However, unlike the depolymerized product obtained by thedeacetylative-deaminative depolymerization method described in thepatent application CN 103214591 A, although the non-reducing terminalsof the 3-elimination depolymerization product described in CN201310099800 are relatively regular, its reducing terminal compositionsare relatively complicated: the reducing terminal residues include both“-D-GalNAc” and L-Fuc-(α1→3) substituted “-4-D-GlcA”. Since thestructure of the reducing terminal is relatively complicated, it istechnically difficult to isolate and obtain the purified oligosaccharidefrom the depolymerized product, and therefore it is generally preferredto directly use the depolymerized product in the form of a mixture.

It is easy for those skilled in the pharmacy to understand that thepurified oligosaccharide has a pure chemical structure and a higherquality control level, and thus may have higher application value.Obviously, for the depolymerized product of natural FG the degree ofregularity of the terminal structure may significantly affect thetechnical feasibility of preparation of purified oligosaccharidecompounds.

Theoretically, the β-eliminative depolymerization method can selectivelycleave the “-D-GalNAc-(β1→4)-D-GlcA-” glycosidic bond, and the reducingterminal of the resulting depolymerized product should be “-D-GalNAc”residue, and the oligosaccharide homologue in the resultingdepolymerized product should generally be composed of 3m monosaccharideresidues. A certain amount of oligosaccharide compound having“-[L-Fuc-(α1→3)]-D-GlcA-” at the reducing terminal is present in theβ-elimination depolymerization product of natural FG disclosed in thepatent application CN 201310099800, which indicates that during theβ-elimination reaction under such conditions, there should be some sidereactions, and in particular, the residue at the reducing terminal ofthe depolymerized product is damaged to some extent.

By further study of the β-elimination reaction conditions of natural FGQthe present inventors have found that using a reducing agent couldreduce the reducing terminal residue of natural FG to its conrespondingalditol, and the carboxylic acid esterification product thereof can besubjected to β-elimination reaction in a basic non-aqueous solventdescribed in patent ZL 201310099800, however, the depolymerized productalso contains some oligosaccharide compounds having “-D-GlcA-” residueat the reducing terminal. According to HPGPC analysis of thedepolymerized product and calculation by area normalization method, theoligosaccharide compound having -D-GlcA at the reducing terminal mayaccount for about 10%-30% of the total amount of the oligosaccharidecompounds, and the result is similar to that of the depolymerizedproduct of natural FG containing hemiacetal structure at the reducingterminal under the same conditions. Studies have shown that thereduction of the reducing terminal residue of natural FG to an alditolgroup does not affect the progress of the β-elimination reaction of theFG carboxyl ester, and does not reduce the destruction of the reducingterminal residue of the depolymerized product, either.

When a reducing agent (for example, sodium borohydride) is directlyadded to the basic non-aqueous solvent described in the β-eliminationreaction of the patent application CN 201310099800, it is found that theβ-elimination reaction of the natural FG carboxyl ester may proceednormally. Unexpectedly, the reducing terminal of the obtained product issubstantially acetylaminogalactitol group (-3-D-GalNAc-ol); while thecontent of the oligosaccharide compound having “-3-D-GlcA (-ol)” residueat the reducing terminal is significantly reduced, and the content maybe less than about 5% or even lower than the HPGPC detection limit,according to the HPGPC area normalization method. Thus, the homologousoligosaccharide compounds in the resulting depolymerized product mayhave a more regular terminal chemical structural feature: all thehomologous oligosaccharide compounds are composed of 3m monosaccharideresidues; the glycosyl at the non-reducing terminal is“L-Fuc-(α1-3)-ΔUA-1-” and the glycosyl group at the reducing terminal is“-3-D-GalNAc-ol”.

By the β-elimination reaction in a basic non-aqueous organic solvent inthe presence of a reducing agent and chromatographic separationtechnique, the inventors first isolated and purified a series ofpurified oligosaccharide compounds with novel chemical structures fromthe β-elimination depolymerization product of FG The purifiedoligosaccharide compounds have a common chemical structural feature: thepurified oligosaccharides are composed of 3m monosaccharide residues,and the non-reducing terminal structure is “L-Fuc-(α1-3)-ΔUA-1-”, andthe reducing terminal glycosyl group is “-3-D-GalNAc-ol”.

The inventors have further studied and found that an oligosaccharidecontaining 3m monosaccharide residues can lose a monosaccharide residuethrough a “peeling reaction” at the reducing terminal, thereby producingan oligosaccharide “containing (3m-1) monosaccharide residues”, and thereducing terminals of such oligosaccharides are all “-D-GlcA”. Throughintensive studies on the β-elimination reaction conditions of FGcarboxyl esters, the inventors have also surprisingly found:

When a small amount of aqueous solution of a strong base (for example,NaOH) is added to the non-aqueous basic reaction solution describedabove, the FG oligosaccharide containing 3m monosaccharide residues andhaving “-3-D-GalNAc” at the reducing end is highly susceptible to the“peeling reaction” and lose the terminal “-D-GalNAc” glycosyl group.Unexpectedly, the oligosaccharide compound (which contains (3m-1)monosaccharide residues) having “-D-GlcA” at the reducing terminalproduced by the “peeling reaction” is “unexpectedly” difficult to have afurther “peeling reaction”. Therefore, by improving the basic treatmentconditions of the FG carboxyl ester in a non-aqueous solvent, after theβ-elimination method cleaves the D-GalNAc-(β1→4)-D-GlcA glycosidic bond,the terminal “-3-D-GalNAc” glycosyl group of the depolymerized productcan be further removed by the “peeling reaction” of the reducingterminal, thereby obtaining the oligosaccharide homologues with noveland regular chemical structural features.

HPGPC chromatographic analysis and NMR structural analysis show that inan anhydrous organic solvent, treating FG carboxyl ester with a strongbase causes “β-elimination depolymerization”, and then adding a smallamount of strong basic aqueous solution to the reaction solution tofurther subject the depolymerized product of the β-eliminationdepolymerization to “peeling reaction”, and the homologousoligosaccharide compounds contained in the depolymerized product mayhave a very regular chemical structure, that is, the homologousoligosaccharide compounds are composed of (3m-1) monosaccharideresidues; the non-reducing terminal glycosyl group of the homologousoligosaccharide compounds is “L-Fuc-(α1-3)-ΔUA-1-”, and the reducingterminal glycosyl group is “-4-D-GlcA” substituted by L-Fuc at the C3position.

The oligosaccharide homologue obtained by the “β-elimination” and“peeling reaction” treatment of natural FG carboxyl ester have moreregular chemical structural features, and thus is easily isolated andpurified to obtain a series of purified oligosaccharide compounds. Thecommon structural feature of the series of purified oligosaccharidecompounds is that all the oligosaccharide compounds contain (3m-1)monosaccharide residues; the non-reducing terminal is“L-Fuc-(α1-3)-ΔUA-1-”; and the reducing terminal is“-4-[Fuc-(α1-3)]-D-GlcA”.

It can be seen from the above that by further improving theβ-elimination conditions of the natural FG carboxyl esters, the presentinvention can obtain a depolymerized product of FG having a more regularstructure (especially a reducing terminal glycosyl structure): one is adepolymerized product having “-D-GalNAc-ol” at the reducing terminal,and the other is a depolymerized product having “-D-GlcA” at thereducing terminal. Since the terminal structure of the depolymerizedproduct is more regular, the present invention first discloses a seriesof purified oligosaccharide compound derived from natural FG which isisolated from such depolymerized products. The present invention furtherdiscloses various series of derivatives of the FG oligosaccharidecompounds by structural modifications of specific chemical groups ofsuch purified oligosaccharides.

Furthermore, by studying the structure-activity relationship onanti-coagulant activity of FG oligosaccharide compounds, inhibitoryactivity against intrinsic factor Xase, and activity of heparin cofactorII (HC-II)-dependent antithrombin (i.e., active coagulation factor IIa),the present inventors also found that:

For FG oligosaccharide homologue with a reducing terminal of“-D-GalNAc-ol” and containing 3m monosaccharide residues, the minimumstructural fragment with potent inhibition against intrinsic factor Xaseis nonasaccharide (NSac); for the FG oligosaccharide homologue with areducing terminal of “-D-GlcA” and containing (3m-1) monosaccharideresidues, the minimum structural fragment with potent inhibition againstintrinsic factor Xase is octasaccharide (OSac); all the purifiedoligosaccharide compounds also have different intensity of HC-IIdependent antithrombin activity and in vitro anticoagulant activity, andhave pharmacological activity of inhibiting arteriovenous thrombosis inpathological models of experimental animals.

Since the purified oligosaccharide compounds of the present inventionand the oligosaccharide derivatives obtained by the structuralmodification thereof have coagulation factor inhibitory activity as wellas significant anticoagulant and antithrombotic activity, theseoligosaccharide compounds have potential application value of preventionand/or treatment for thrombotic diseases.

In general, the present invention first discloses a technical method ofobtaining natural FG depolymerized product with more regular chemicalstructure by “β-elimination depolymerization” or “β-eliminationdepolymerization and terminal peeling reaction” and a FG oligosaccharidehomologue with a homogenous structure obtained by such method. Thepresent invention also first discloses a purified FG oligosaccharidecompound having unsaturated hexuronic acid residue structure at thenon-reducing terminal, a structurally modified derivative thereof, and amixture thereof. Since the oligosaccharide compound has anticoagulantand antithrombotic activity, the present invention also discloses theuse of the oligosaccharide compound and a mixture thereof for thepreparation of a medicament for the prevention and/or treatment ofthrombotic diseases.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a purifiedoligosaccharide compound having anticoagulant and antithromboticactivity, a method for preparing the same, a pharmaceutical compositioncomprising the purified oligosaccharide compound or an oligosaccharidemixture and a pharmaceutically acceptable salt thereof, and use of theoligosaccharide compound, the oligosaccharide mixture and thepharmaceutical composition thereof for the preparation of a medicamentfor the prevention and/or treatment of thrombotic diseases.

The present invention first provides an oligosaccharide compound havingantithrombotic activity, particularly an activity of inhibitingintrinsic coagulation factor Xase, and a pharmaceutically acceptablesalt thereof. The oligosaccharide compound has a general structurerepresented by Formula (I):

in Formula (I),

R₁, R₂, R₃, R₄, R₅ are optionally and independently —H or —SO₃H;

R₆ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbongroup or a C7-C12 aryl group;

R₇ is optionally —H, —SO₃, C₂-C₅ acyl;

R₈ is optionally a group represented by Formula (II), Formula (III) orFormula (IV):

in Formula (II), Formula (III) and Formula (IV),

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are all defined as above;

R₉ and R₁₀ are optionally —H, a substituted or unsubstituted C1-C6hydrocarbon group or a C7-C12 aryl group;

R₁₁ is optionally —NHR₁₂ or —OR₁₃, wherein, R₁₂ and R₁₃ are optionally—H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12aryl group;

n is optionally 0 or a natural number of 1˜8.

The oligosaccharide compound having the general structure represented byFormula (I) according to the present invention means a “purifiedoligosaccharide compound”. In general, the “purified oligosaccharidecompound” has a purity of no less than 95%. For example, by analyzingwith analytical high-performance gel chromatography (HPGPC), such asAgilent high performance liquid chromatography and gel column, anddetecting with a universal differential detector (RID), the purifiedoligosaccharide compound generally has a purity of no less than 95%,which is calculated according to the area normalization method.

Among the oligosaccharide compounds of the structure of Formula (I) ofthe present invention, a preferred oligosaccharide compound is thecompound in which R₈ is a group represented by Formula (II), that is,the oligosaccharide compound has the general structure represented byFormula (V):

in Formula (V),

R₁, R₂, R₃, R₄, R₅ are optionally and independently —H or —SO₃H;

R₆ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbongroup or a C7-C12 aryl group;

R₇ is optionally —H, —SO₃H, C2-C5 acyl;

R₉ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbongroup or a C7-C12 aryl group;

n is optionally 0 or a natural number of 1˜8.

In a more preferred compound of Formula (V), R₁═—H, R₂═R₃═R₄═R₅═—SO₃H;

In another more preferred compound of Formula (V), R₁═R₃═R₄═R₅═—SO₃H;R₂═—H.

Among the oligosaccharide compounds of Formula (I) of the presentinvention, another preferred oligosaccharide compound is the compound inwhich R₈ is a group represented by Formula (III), that is, theoligosaccharide compound has the general structure represented byFormula (VI):

in Formula (VI),

R₁, R₂, R₃, R₄, R₅ are optionally and independently —H or —SO₃H;

R₆ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbongroup or a C7-C12 aryl group;

R₇ is optionally —H, —SO₃H, C2-C5 acyl;

R₁₀ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbongroup or a C7-C12 aryl group; and

n is optionally 0 or a natural number of 1˜8.

Similarly, in a more preferred compound of Formula (VI), R₁═—H;R₂═R₃═R₄═R₅═—SO₃H;

In another more preferred compound of Formula (VI), R₁═R₃═R₄═R₅═—SO₃H;R₂═—H.

In the oligosaccharide compound of Formula (I) of the present invention,another preferred oligosaccharide compound is the compound in which R₈is a group represented by Formula (VII), that is, the oligosaccharidecompound has the general structure represented by Formula (VII):

in Formula (VII),

R₁, R₂, R₃, R₄, R₅ are optionally and independently —H or —SO₃H;

R₆ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbongroup or a C7-C12 aryl group;

R₇ is optionally —H, —SO₃H, C2-C5 aryl group;

R₁₁ is optionally —NHR₁₂ or —OR₁₃, wherein, R₁₂ and R₁₃ are optionally—H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12aryl group; and

n is optionally 0 or a natural number of 1˜8.

Similarly, in a more preferred compound of Formula (VII), R₁═—H;R₂═R₃═R₄═R₅═—SO₃H;

In another more preferred compound of Formula (VII), R₁═R₃═R₄═R₅═—SO₃H;R₂═—H.

It will be readily understood by those skilled in the art that thetechnical difficulty in isolating and purifying the oligosaccharidecompound of the present invention may increase as the degree ofpolymerization of the oligosaccharide increases. Therefore, among theoligosaccharide compounds of the structure represented by the aboveFormula (I), (V), (VI) or (VII) of the present invention, preferredoligosaccharide compounds are those in which n is optionally 1, 2, 3 or4.

The purified oligosaccharide compound of the present invention hassulfate substituents and/or free carboxyl groups, and thus can becombined with a pharmaceutically acceptable inorganic and/or organic ionto form a salt. In general, the pharmaceutically acceptable salt of theoligosaccharide compound of the present invention may be optionally analkali metal salt, an alkaline earth metal salt or an organic ammoniumsalt.

Preferred pharmaceutically acceptable salt of the oligosaccharidecompound of the present invention is a sodium salt, a potassium salt ora calcium salt.

It will be readily understood by those skilled in the art that when thepurified oligosaccharide compounds of the present invention in the formof homologues, such as homologues of the compound of Formula (V), orhomologues of the compound of Formula (VI) or homologues of the compoundof Formula (VII) described above, are mixed, a mixture of homologousoligosaccharide compounds having a specific structure type may beobtained. In particular, according to the preparation method of thecompound of the present invention described later in the specification,the present invention may also obtain a FG oligosaccharide mixture inthe form of homologues having more regular chemical structure(especially a reducing terminal glycosyl structure type) by a specifictechnical method.

Thus, the present invention also provides an oligosaccharide mixturehaving antithrombotic activity, particularly an activity of inhibitingintrinsic factor tenase, and a pharmaceutically acceptable salt thereof.The oligosaccharide mixture is composed of homologues of the aboveoligosaccharide compound of Formula (I); and R₈ of the oligosaccharidecompound of Formula (I) in the oligosaccharide mixture is a grouprepresented by Formula (II), or a group represented by (III), or a grouprepresented by Formula (IV). Specifically, based on the molar ratio, theoligosaccharide compound in which R₈ is the group represented by Formula(II) accounts for not less than 95% in the mixture, or theoligosaccharide compound in which R₈ is the group represented by Formula(III) accounts for not less than 95% in the mixture; or theoligosaccharide compound in which R₈ is the group represented by Formula(IV) accounts for not less than 95% in the mixture.

In the oligosaccharide mixture of the present invention, a preferredoligosaccharide mixture is a mixture of homologous oligosaccharidecompounds having the general structure represented by the above Formula(V). A more preferred oligosaccharide mixture of the present inventionis a mixture of homologous oligosaccharide compounds of the structurerepresented by Formula (V), in which R₁═—H; R₂═R₃═R₄═R₅═—SO₃H; inanother more preferred mixture of homologous oligosaccharide compoundsof the structure represented by Formula (V), R₁═R₃═R₄═R₅═—SO₃H; R₂═—H.

In the oligosaccharide mixture of the present invention, anotherpreferred oligosaccharide mixture is a mixture of homologousoligosaccharide compounds having the general structure represented byFormula (VI). Similarly, in more preferred mixture of homologousoligosaccharide compounds of the structure represented by Formula (VI),R₁═—H; R₂═R₃═R₄═R₅═—SO₃H; in another more preferred mixture ofhomologous oligosaccharide compounds of the structure represented byFormula (VI), R₁═R₃═R₄═R₅═—SO₃H; R₂═—H.

In the oligosaccharide mixture of the present invention, anotherpreferred oligosaccharide mixture is a mixture of homologousoligosaccharide compounds having the general structure represented byFormula (VII). Similarly, in a more preferred mixture of homologousoligosaccharide compounds of the structure represented by Formula (VII),R₁═—H; R₂═R₃═R₄═R₅═—SO₃H; and in another more preferred mixture ofhomologous oligosaccharide compounds of the structure represented byFormula (VII), R₁═R₃═R₄═R₅═—SO₃H; R₂═—H.

For the oligosaccharide compound and the oligosaccharide mixture of thepresent invention described above, the present invention still furtherprovides a method for preparing the compound and the mixture.

First, the present invention provides a preparation method of theoligosaccharide compound of the structure represented by Formula (I) anda pharmaceutically acceptable salt thereof. In the preparation method,fucosylated glycosaminoglycan (FG) derived from an echinoderm is used asa starting material of the reaction, and optionally is depolymerized bythe following method:

Esterifying the FG carboxyl group and subjecting the FG carboxylate to“β-elimination reaction” and depolymerization in an anhydrous organicsolvent in the presence of a strong base and a reducing agent, andreducing the -D-acetylaminogalactosyl (-D-GalNAc) at the reducingterminal of the depolymerized product to an alditol (-D-GalNAc-ol),thereby obtaining a mixture of homologous oligosaccharide compounds;

Esterifying the FG carboxyl group and subjecting the FG carboxylate to“β-elimination reaction” and depolymerization in an anhydrous organicsolvent in the presence of a strong base, followed by subjecting thedepolymerized product to terminal “peeling reaction” by adding a basicaqueous solution to lose the -D-GalNAc at the reducing terminal, andobtain a mixture of homologous oligosaccharide compounds having-D-glucuronic acid (-D-GlcA) at the reducing terminal.

The mixture of homologous oligosaccharide compounds obtained by the“β-elimination depolymerization and terminal reduction” or“β-elimination depolymerization and peeling reaction” is isolated andpurified and optionally structurally modified to obtain the desiredpurified oligosaccharide compound.

In particular, the method for preparing an oligosaccharide compound ofthe present invention is that for preparing an oligosaccharide compoundhaving the structure represented by Formula (I) and having R₈ as thegroup represented by the above Formula (II). The named “oligosaccharidecompound having the structure represented by Formula (I) and having R₈as the group represented by Formula (II)” is substantially equivalent tothe oligosaccharide compound of the structure represented by Formula (V)defined above.

The preparation method of the oligosaccharide compound of the structurerepresented by Formula (V) is a “β-elimination depolymerization+terminalreduction” method. The method comprises: in the presence of a strongbase and a reducing agent in an anhydrous organic solvent, subjectingthe carboxylated FG to a “β-elimination reaction” to cleave its“D-GalNAc-(β1→4)-GlcA” glycosidic bond, and reducing the reducingterminal D-GalNAc of the depolymerized product with a reducing agent to-D-GalNAc-ol, thereby obtaining a mixture of homologous oligosaccharidecompounds with relatively regular terminal structure, followed byisolating and purifying, and optional structural modifying the specificsubstituent to obtain the desired purified oligosaccharide compound. Thespecific steps comprise:

(a) converting natural FG into a quaternary ammonium salt form, andcompletely or partially converting the carboxyl groups on D-GlcA in theFG quaternary ammonium salt into a carboxyl ester in an organic solvent;

(b) in an organic solvent having a reducing agent, treating thecarboxylated FG quaternary ammonium salt of the step (a) with a strongbase to cause β-elimination depolymerization, and reducing the -D-GalNAcat the reducing terminal of the depolymerized product to -D-GalNAc-ol,thereby obtaining a mixture of homologous oligosaccharide compounds witha relatively regular terminal structure;

(c) converting the mixture of homologous oligosaccharide compoundsobtained in the step (b) into an alkali metal salt form, and in anaqueous solution, subjecting the carboxylate of the homologousoligosaccharide compound to basic hydrolysis, to obtain a mixture ofhomologous oligosaccharide compounds containing a free carboxyl group;

(d) isolating and purifying the homologous oligosaccharide compound bychromatography from the oligosaccharide mixture obtained in the step(c);

(e) optionally subjecting the purified oligosaccharide compound obtainedin the step (d) to a further structural modification.

The steps (a)˜(e) are as shown in the route scheme 1.

In Scheme 1:

Natural FG 1 is a natural FG derived from an echinoderm, which is amixture of series of homologous polysaccharides;

FG ammonium salt 2 is a FG in the form of a quaternary ammonium salt;

FG ester ammonium salt 3 is a product in which part of or all thecarboxyl groups on D-GlcA in the FG are esterified, which is present inthe form of a quaternary ammonium salt;

dFG ester ammonium salt 4 is a depolymerized product formed byβ-elimination reaction of the FG carboxylate, and the reducing terminalglycosyl-D-GalNAc could be reduced by the reducing agent present in thereaction solution to form a reduced dFG ester ammonium salt 5;

Reduced dFG 6 is a carboxylate hydrolyzate of 5 as an alkali metal salt;depolymerized products 4, 5 and 6 are all a mixture of homologousoligosaccharide compounds;

Purified oligosaccharide 7 is a purified oligosaccharide obtained byisolating from the depolymerized product 6; and the purifiedoligosaccharide 8 is a purified oligosaccharide obtained by optionallysubjecting 7 to a substituent structural modification;

In the chemical structure of Scheme 1, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₉and n are all defined as in the above Formula (I); x is a natural numberhaving a mean value in the range of about 40˜80; y is a natural numberin the range of about 0˜15; —COOX is a carboxylate or a quaternaryammonium salt of a carboxylic acid.

In Scheme 1, it can be seen from the chemical structure of natural FGthat it has a chondroitin sulfate-like backbone structure and hassulfated L-Fuc side chain substituents. In general, natural FG can beunderstood as a polysaccharide compound formed by sequential linkage ofthe“trisaccharide structural units”{-4)-[L-FucS-(α1-3)]-D-GlcA-(β1-3)-D-GalNAcS-(β1-} (wherein, FucS andGalNAcS represent sulfated Fuc and sulfated GalNAc, respectively). Ingeneral, natural FG typically contains a mean of about 40 to 80 of suchtrisaccharide structural units (approximately, the mean value of x is inthe range of about 40-80).

The form in which the natural FG salt is present depends on the route ofits extraction and purification. In general, the FG is present in theform of an alkali metal or alkaline earth metal salt (such as a sodiumsalt, a potassium salt or a calcium salt thereof). In order to achievethe chemical reaction in the organic solvent in the subsequent steps,natural FG is converted into a quaternary ammonium salt form in the step(a) (2 in Scheme 1).

The conversion of natural FG into a quaternary ammonium salt canoptionally be carried out using techniques well known in the art. Forexample, the conversion into quaternary ammonium salt can be performedby quaternary ammonium salt precipitation method, which comprises addingan excess of an organic ammonium salt compound to an aqueous solution ofan alkali metal or alkaline earth metal salt of FG thereby forming awater-insoluble FG quaternary ammonium salt that can be easilyprecipitated from the aqueous solution; in addition, an alkali metalsalt or an alkaline earth metal salt of FG can also be exchanged into anH-form FG using an ion exchange resin, followed by neutralization of theH-form FG with a basic organic ammonium to obtain a FG quaternaryammonium salt.

As shown in Scheme 1, the step (a2) comprises convering all or part ofcarboxyl groups on the D-GlcA residue in the FG quaternary ammonium salt(2) into a carboxylate (3). The purpose of the carboxyl esterificationreaction of FG is to make it susceptible to the β-elimination reaction.The GlcA in the form of a carboxyl group is less likely to undergo aβ-elimination depolymerization reaction, and its carboxylate issusceptible to the β-elimination reaction due to the electronic effectof the ester group.

Generally, the carboxyl esterification reaction of GlcA in the FGcomprises: in an organic solvent such as dimethylformamide (DMF) or amixed solvent of DMF and a lower alcohol, a ketone and/or an ether,reacting the carboxyl group on GlcA in the FG with a stoichiometricamount of a halogenated hydrocarbon, to easily obtain a desired FGcarboxylate with different degrees of esterification. The degree ofesterification of the FG carboxylate means the ratio of the number ofmoles of the carboxylate group formed after the esterification reactionto the number of moles of the free carboxyl group before theesterification reaction; the halogenated hydrocarbon may optionally beand is not limited to: a C1-C6 linear or branched, saturated orunsaturated, substituted or unsubstituted aliphatic hydrocarbon group;or a substituted or unsubstituted C7-C12 aromatic hydrocarbon group andso on. The present applicant discloses a method for the preparation of aFG carboxylate derivative in another invention patent application CN201110318704.X, which is incorporated herein by reference in itsentirety.

The step (b) shown in Scheme 1 comprises subjecting the FG carboxylateto β-elimination depolymerization (b1) to obtain the depolymerizedproduct 4, and reducing the reducing terminal D-GalNAc of thedepolymerized product 4 by a reducing agent to -D-GalNAc-ol (b2), and toobtain the depolymerized product 5.

As described above, CN 201310099800 discloses a β-eliminationdepolymerization method of natural FG The method can obtain adepolymerized product having unsaturated ΔUA at the non-reducingterminal, but the structural type at the reducing terminal of thedepolymerized product is relatively complicated: the glycosyl group atthe reducing terminal includes both “-D-GalNAc” and L-Fuc substituted“-D-GlcA”. Since the structure of the reducing terminal is complicated,it is difficult to isolate and obtain a purified oligosaccharide fromthe depolymerized product, and therefore it is generally preferred todirectly use the depolymerized product in the form of a mixture.Theoretically, the β-elimination depolymerization method can selectivelycleave the “D-GalNAc-(β1→4)-D-GlcA” glycosidic bond, and the resultingdepolymerized product has a “-D-GalNAc” residue at the reducingterminal. Some amount of oligosaccharide compound having “-D-GlcA” atthe reducing terminal is present in the depolymerized product of naturalFG prepared by the β-elimination depolymerization method described in CN201310099800, indicating that there are still some side reactions underthe reaction conditions. In particular, the glycosyl group at thereducing terminal of some amount of depolymerized product is destroyed.The patent application CN 201310099800 is incorporated herein byreference in its entirety.

It will be readily understood by those skilled in the art that thepurified oligosaccharide has a pure structure and a higher level ofquality control, and thus may have higher application value. Throughfurther studies on the β-elimination reaction conditions of natural FGSthe present inventors have surprisingly found that:

The glycosyl group at the reducing terminal of the natural FG is reducedto an alditol by a reducing agent such as sodium borohydride, and thecarboxylated product can undergo a β-elimination reaction in a basicnon-aqueous solvent, but according to the HPGPC spectrum analysis of thedepolymerized product, the oligosaccharide compound having -D-GlcA atthe reducing terminal may account for about 10%˜30% of the total amountof the oligosaccharide compound (area normalization method), and theresult is similar to that of the depolymerized product of natural FGhaving unreduced reducing terminal under the same conditions. Thisresult indicates that the terminal reduction does not affect theprogress of the β-elimination reaction of the FG carboxylate.

Further studies have shown that when a reducing agent (such as sodiumborohydride) is directly added to the basic non-aqueous solvent, and theβ-elimination reaction of the natural FG carboxylate may also be carriedout normally. Unexpectedly, the reducing terminal of the obtainedproduct is substantially -3-D-GalNAc-ol, while the content of theoligosaccharide compound having -3-D-GlcA-ol at the reducing terminal isvery small (the content may be less than about 5%, even below the HPGPCdetection limit). This result indicates that the terminal reduction ofthe depolymerized product can effectively avoid the destruction of thereducing terminal glycosyl group caused under basic conditions, therebyachieving the relatively regular structure of the reducing terminal ofthe depolymerized product.

It will be readily understood by those skilled in the art that due tothe presence of a reducing agent (such as sodium borohydride) in thereaction solution, the terminal glycosyl group of the depolymerizedproduct obtained from the β-elimination depolymerization can be rapidlyreduced to an alditol. On the one hand, the reduction of the reducingterminal glycosyl group to the alditol does not affect the furtherβ-elimination reaction of the hexuronic acid ester; on the other hand,after the terminal glycosyl group in the depolymerized product isreduced to the alditol, the destruction and degradation of the reducingterminal glycosyl group under basic conditions can be effectivelyavoided. Therefore, the β-elimination depolymerization of the FGcarboxylate in the presence of a reducing agent can obtain adepolymerized product with a more regular chemical structure.

The “more regular chemical structure” means that: (1) the homologousoligosaccharide compound contained in the depolymerized product iscomposed of 3m monosaccharide residues; (2) the non-reducing terminalglycosyl group of the homologous oligosaccharide compound is“L-Fuc-(α1-3)-ΔUA-1-”, and the reducing terminal glycosyl group is“-3-D-GalNAc-ol”.

Thus, the technical feature of the step (b) is that the β-eliminationdepolymerization reaction is carried out in the presence of a reducingagent, and the β-elimination reaction condition means that the FGcarboxylate is treated in a non-aqueous solvent with a strong base.Since the reaction solution for the FG carboxylic acid esterification inthe step (a) is a non-aqueous solvent, after the carboxylic acidesterification reaction is completed, the reaction solution will bedirectly used for the β-elimination depolymerization reaction of thestep (b) without further treatment.

In the step (b), the reducing agents are those that can reduce thereducing terminal glycosyl group to an alditol, such as sodiumborohydride; the amount of the reducing agent is related to the amountof the depolymerized product formed. Those skilled in the art willappreciate that in order to ensure the yield and structural uniformityof the depolymerized product, a stoichiometric excess of reducing agentshould generally be employed in the reaction. On the other hand, thestrong base in the step (b) may be optionally a lower sodium alkoxide, adiazabicyclo ring or the like.

The step (c) shown in Scheme 1 comprises converting the homologousoligosaccharide mixture 5 obtained by β-elimination depolymerization ofFG to an alkali metal salt, which comprises optionally adding asaturated aqueous solution of an inorganic salt (such as sodiumchloride) to the reaction solution. The basic hydrolysis of thecarboxylate of the homologous oligosaccharide compound may be generallycarried out by treatment with an aqueous solution of an inorganic base(for example, 0.05 M˜1 M NaOH or KOH), thereby obtaining a homologousoligosaccharide mixture 6 containing a free carboxyl group.

The step (d) shown in Scheme 1 comprises isolating and purifying theoligosaccharide mixture to obtain a series of purified oligosaccharidecompounds 7. In general, the isolation and purification of theoligosaccharide compound by chromatography as described in the step (d)means that the oligosaccharide compound is purified by gelchromatography and/or ion exchange chromatography, and the gelchromatography and/or ion exchange chromatography is a method well knownto those skilled in the art. In addition, the gel chromatography and/orion exchange chromatography may optionally be combined with a technicalmethod such as ultrafiltration or salting out method to increase theefficiency of the isolation and purification.

The step (e) shown in Scheme 1 comprises optionally subjecting theoligosaccharide compound 7 obtained in the step (d) to a furtherstructural modification, thereby obtaining the oligosaccharide compound8. The compound 8 is a oligosaccharide compound of Formula (I) in whichR₈ is a group represented by Formula (II), which is equivalent to theoligosaccharide compound represented by the above Formula (V). Wherein:

The oligosaccharide compound 7 is subjected to quaternary ammonium saltconversion, and then reacted with a halogenated hydrocarbon in anorganic solvent by a conventional method in the art, to easily obtain anoligosaccharide compound of Formula (V) in which R₆ is a C1-C6 aliphatichydrocarbon group or a C7-C12 aryl group.

By the hydrazinolysis method described in CN 103214591 A and relatedliterature (Proc Natl Acad Sci USA. 2015; 112(27): 8284-9), the acetylgroup on D-GalNAc in oligosaccharide compound 7 can be removed, toobtain a deacetylated oligosaccharide compound, namely, anoligosaccharide compound of Formula (V) in which R₇ is —H. Thedeacetylated oligosaccharide compound can be reacted with an acidanhydride or Et₃N.SO₃ to obtain an N-reacylated or resulfatedoligosaccharide compound, namely, an oligosaccharide of Formula (V) inwhich R₇ is a C2-C5 acyl group or —SO₃H.

Further, the alcoholic hydroxyl at the C1 position of -D-GalNAc-ol atthe reducing terminal of the oligosaccharide compound 7 may optionallybe reacted with an alcohol compound under acidic conditions to form aterminal alkylation product. It will be readily understood by thoseskilled in the art that a compound of Formula (V) in which R₉ is asubstituted or unsubstituted C1-C6 aliphatic hydrocarbon group or aC7-C12 aryl group can be obtained by the alkylation reaction.

Obviously, by using the above structural modification method incombination, the oligosaccharide compound represented by Formula (V)with various specific structures defined by the present invention can beobtained.

For the preparation method shown in Scheme 1, a preferred embodiment is:

In the step (a), the FG quaternary ammonium salt isN,N-dimethyl-N-[2-[2-[4(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethylbenzammonium salt, namely benzethonium salt; the organic solvent is DMFor a DMF-ethanol mixture; the carboxylate is a benzyl ester; and the“complete or partially conversion into carboxylate” means that thedegree of carboxyl esterification of the mixture 3 is in the range fromabout 30% to about 100%.

In the step (b), the organic solvent is DMF or a DMF-ethanol mixture;the reducing agent is sodium borohydride; and the strong base is sodiumethoxide.

In the step (c), the conversion of the quaternary ammonium salt mixtureinto an alkali metal salt comprises adding a saturated aqueous solutionof sodium chloride to the reaction solution to convert the obtainedoligosaccharide homologue 5 into a sodium salt form; the basichydrolysis in the aqueous solution means that the carboxylate ofoligosaccharide compound is hydrolyzed in NaOH aqueous solution with aconcentration of 0.05 M˜1 M.

In the step (d), the chromatography includes, but is not limited to, gelchromatography and/or ion exchange chromatography;

In the step (e), the further structural modification includes, but isnot limited to, carboxyl esterification of D-glucuronic acid group(GlcA) and unsaturated hexuronic acid group (ΔUA) in the oligosaccharidecompound; deacetylation and optional reacylation or resulfation ofD-acetylgalactosamine group (D-GalNAc); alkylation of alditol at thereducing terminal (D-GalNAc-ol).

It is known that there may be differences in the sulfated form ofnatural FG from different species sources. Among them, the reportedsulfated forms of the FG side chain L-Fuc include 2,4-disulfate(L-Fuc_(2S4S)), 3,4-disulfate (L-Fuc_(3S4S)), 3-sulfate (L-Fuc_(3S)) and4-sulfate (L-Fuc_(4S)) and no sulfate group substitution; the reportedsulfated forms of D-GalNAc in the backbone include 4,6-disulfate(D-GalNAc_(4S6S)), 4-sulfate (D-GalNAc_(4S)), 6-sulfate (D-GalNAc_(6S))and no sulfate group substitution. Also, some natural FGs may havedifferent sulfated forms of L-FucS and/or D-GalNAcS, while other naturalFGs contain a relatively regular and single sulphated form of L-FucSand/or D-GalNAcS (refer to: Pomin V H. Mar Drugs. 2014, 12, 232-54).

It can be seen from the preparation method of the oligosaccharidecompound of Formula (V) that all the steps do not affect the stabilityof the sulfate group on the glycosyl group, and thus the sulfated formof the obtained oligosaccharide compound depends on the sulfated form ofthe natural FG Obviously, for the natural FG having a relatively regularsulfated form, the type of the oligosaccharide compound in itsβ-elimination depolymerization product is relatively small, and theoligosaccharide compounds having the same polymerization degree have thesame chemical structure, and thus the purified oligosaccharide compoundof the present invention can be easily prepared.

For example, in the purified natural FG extracted from the body wall ofechinoderms such as Stichopus variegatus, Bohadschia argus, andStichopus monotuberculatus, the side chain fucosyl group is mainlyL-Fuc_(2S4S), and the hexosamine in its main chain is mainlyD-GalNAc_(4S6S). Therefore, these natural FGs are suitable for thepreparation of the oligosaccharide compound of Formula (VIII) and apharmaceutically acceptable salt thereof, and the compound issubstantially an oligosaccharide compound of Formula (V) in whichR₁═R₃═R₄═R₅═—SO₃H, and R₂═—H.

In Formula (VIII), R₆, R₇ and R₉ are as defined above.

In the natural FG extracted from the body wall of echinoderms such asHolothuria scabra, Holothuria fuscopunctata, Stichopus horrens, andPearsonotheia graeffei, the side chain fucosyl group is mainlyL-Fuc_(3S4S), and the hexosamine in its main chain is mainlyD-GalNAc_(4S6S). Therefore, these natural FGs are suitable for thepreparation of the oligosaccharide compound of Formula (IX) and apharmaceutically acceptable salt thereof, and the compound issubstantially an oligosaccharide compound of Formula (V) in which R₁═—H,and R₂═R₃═R₄═R₅═—SO₃H.

In Formula (IX), R₆, R₇ and R₉ are as defined above.

Another preparation method of the oligosaccharide compound of thepresent invention is a method of the preparation of an oligosaccharidecompound having the structure represented by Formula (I) and having R₈as a group represented by the above Formula (III) or (IV). The named“oligosaccharide compound having the structure represented by Formula(I) and having R₈ as a group represented by the above Formula (III) or(IV)” is substantially equivalent to an oligosaccharide compound ofFormula (VI) or Formula (VII) as defined above. The preparation methodcomprises “β-elimination depolymerization+peeling reaction”: subjectingthe carboxylated natural FG to β-elimination depolymerization in anorganic solvent in the absence of reducing agent, followed by peelingreaction to make the FG depolymerized product lose the reducing terminalD-GalNAc residue, thereby obtaining a mixture of homologousoligosaccharide compounds having -D-GlcA at the reducing terminal. Themethod comprise the specific steps of:

(a) converting natural FG into a quaternary ammonium salt form, followedby completely or partially converting the carboxyl group on D-GlcA inthe FG quaternary ammonium salt into a carboxylate in an organicsolvent;

(b) in an anhydrous organic solvent, treating the FG carboxylate with astrong base to cause β-elimination depolymerization, and then by addinga small amount of aqueous solution of a strong base, subjecting the FGdepolymerized product to further “peeling reaction” to lose the-D-GalNAc residue at the reducing terminal, thereby obtaining a mixtureof homologous oligosaccharide compounds having -D-GlcA at the reducingterminal;

(c) converting the oligosaccharide mixture obtained in the step (b) intoan alkali metal salt, followed by subjecting the carboxylate of thehomologous oligosaccharide compounds to basic hydrolysis in an aqueoussolution, to obtain a mixture of the homologous oligosaccharidecompounds containing a free carboxyl group;

(d) isolating and purifying the oligosaccharide compound in theoligosaccharide mixture of the step (c) by chromatography;

(e) optionally, subjecting a further structural modification to thepurified oligosaccharide compound obtained in the step (d).

The products treated in the steps (a) to (e) of the method are as shownin Scheme 2:

Scheme 2

In Scheme 2:

Natural FG 1, FG ammonium salt 2, FG ester ammonium salt 3 and dFG esterammonium salt 4 are all defined as in Scheme 1;

dFG ester ammonium salt 5 is a depolymerized product having -D-GlcA atthe reducing terminal, which is formed by subjecting the depolymerizedproduct 4 to “peeling reaction” to lose the terminal -D-GalNAc. dFG 6 isa hydrolyzate of carboxylate group of 5, which is present in the form ofan alkali metal salt. Depolymerized products 4, 5 and 6 are all amixture of homologous oligosaccharide compounds;

Purified oligosaccharide 7 is a purified oligosaccharide obtained byisolation from the depolymerized product 6; and purifiedoligosaccharides 8 and 9 are purified oligosaccharides obtained bysubjecting 7 to an optional substituent structural modification.

In the chemical structure described in Scheme 2, R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₁₀, R₁₁ and n are defined as in Formula (I) above; x, y and —COOXare defined as in Scheme 1.

The quaternary ammonium salt conversion of the natural FG and thecarboxyl esterification of the FG shown in the step (a) of Scheme 2 arethe same as the method described in above-mentioned Scheme 1.

The step (b) shown in Scheme 2 comprises subjecting the FG carboxylateto β-elimination depolymerization (b1) to form the depolymerizatedproduct 4 (containing a small amount of product 5), followed by “peelingreaction” to remove the D-GalNAc residue (b2) at the reducing terminaland obtain the depolymerized product 5.

As described above, in the β-elimination depolymerization product of thenatural FG disclosed in CN 201310099800, both oligosaccharide compoundhaving D-GalNAc at the reducing terminal and some amount ofoligosaccharide compound having “-D-GlcA-” at the reducing terminal arepresent, which may be related to the destruction of the reducingterminal glycosyl group of the β-elimination depolymerization product.Since the structure of the reducing terminal is relatively complicated,it is difficult to isolate and purify an oligosaccharide from thedepolymerized product.

It is known to those skilled in the art that a “peeling reaction” understrong base conditions can cause some polysaccharide compounds (such ascellulose) to lose the monosaccharide residues at the reducing terminalone by one (Whistler R L, et al. Alkaline Degradation ofPolysaccharides. Advances in Carbohydrate Chemistry. 1958, 13: 289-329).In fact, the present inventors have found that when natural FG isdepolymerized under the conditions described in CN 201310099800, theresulting product has some amount of by-products containing unsaturatedsmall molecule compounds that are similar to the reported products ofthe destruction of reducing terminal glycosyl group in the peelingreaction. In view of the fact that the β-elimination reaction of naturalFG disclosed in CN 201310099800 is carried out under strong baseconditions, it can be inferred that the formation of an oligosaccharidecompound having “-D-GlcA-” at the reducing terminal in the depolymerizedproduct may be related to the “peeling reaction” of the depolymerizedproduct.

It will be understood by those skilled in the art that the reduction ofthe terminal of the depolymerized product into an alditol by addition ofa reducing agent to the reaction solution of β-eliminationdepolymerization is helpful to avoid the occurrence of “peelingreaction”; on the other hand, it is possible to obtain a series of FGoligosaccharides having “-D-GlcA” at the reducing terminal by “peelingreaction” of β-elimination depolymerization product of FG undercontrolled conditions. Accordingly, the present inventors havesurprisingly found through further studies on the β-elimination reactionconditions of the FG carboxylate:

(1) when a small amount of aqueous solution of a strong base (such asNaOH) is added to the basic non-aqueous solution mentioned above, the FGoligosaccharide having “-D-GalNAc” at the reducing terminal is highlysusceptible to “peeling reaction” and loses the glycosyl group at thereducing terminal; unexpectedly, the FG oligosaccharide compound having“-D-GlcA” at the reducing terminal is difficult to undergo the similar“peeling reaction”.

(2) HPGPC analysis and NMR structural analysis show that when the FGcarboxylate is treated with a strong base in an anhydrous organicsolvent to cause “β-elimination depolymerization”, and then a smallamount of aqueous solution of a strong base is added to the reactionsolution to further cause “peeling reaction” of the β-eliminationdepolymerization product, the “homologous oligosaccharide compound”contained in the depolymerized product have a very regular chemicalstructure, that is, the homologous oligosaccharide compound is composedof (3m-1) monosaccharide residues; the non-reducing glycosyl group ofthe homologous oligosaccharide compound is “L-Fuc-(α1-3)-ΔUA-1-”, andthe reducing terminal glycosyl group is “-4-D-GlcA” substituted byL-FucS at the C3 position.

(3) The oligosaccharide homologues obtained by β-eliminationdepolymerization and peeling reaction of the natural FG carboxylate canbe further isolated and purified to obtain a series of “purifiedoligosaccharide compounds”. These purified oligosaccharide compoundshave a common chemical structural feature: the oligosaccharide compoundscontain (3m-1) monosaccharide residues; the non-reducing terminal is“L-Fuc-(α1-3)-ΔUA-1-”, and the reducing terminal is“-4-[L-Fuc-(α1-3)]-D-GlcA”.

Therefore, the step (b) first comprises treating the FG carboxylate witha strong base in a non-aqueous solvent in absence of a reducing agent tocause β-elimination depolymerization (b1 of Scheme 2) to obtain thedepolymerized product 4 (which may contain a small amount ofoligosaccharide 5). Similarly, since the reaction solvent of the FGcarboxyl esterification is a non-aqueous solvent, the reaction solutionis directly used for the β-elimination depolymerization described in thestep (b) without further treatment. In general, the strong base in thestep (b) may be optionally a lower sodium alkoxide, a diazabicyclo ringor the like.

The step (b) shown in Scheme 2 further comprises converting thedepolymerized product 4 into a depolymerized product 5 by further“peeling reaction” (b2 of Scheme 2), which is performed by adding asmall amount of aqueous solution of a strong base to the β-eliminationreaction solution. Generally, the aqueous solution of the strong basemay be optionally 0.25 M˜2 M NaOH, KOH or a saturated Ca(OH)₂ aqueoussolution; the “small amount” of aqueous solution of a strong base meansthat the aqueous solution of the strong base is equivalent to about ⅕˜1/10 of the total volume of the reaction solution.

The step (c) shown in Scheme 2 comprises converting the depolymerizedproduct 5 into an alkali metal salt and hydrolyze the carboxylate, thetechnical method of which is the same as the method described in Scheme1;

Similarly, as shown in the step (d) of Scheme 2, a mixture ofoligosaccharide compounds 6 is isolated and purified to obtain a seriesof purified oligosaccharide compounds 7. In general, the purificationmethod refers to gel chromatography and/or ion exchange chromatography,and may optionally be combined with technical methods such asultrafiltration, salting out method to improve the efficiency ofisolation and purification.

Similarly, the step (e) comprises optionally subjecting a furtherstructural modification to the oligosaccharide compound 7 obtained inthe step (d), thereby obtaining a purified oligosaccharide compound 8 or9. Wherein:

The oligosaccharide compound 8 is substantially equivalent to theoligosaccharide compound of Formula (VI) described above, and thecompound 9 is substantially equivalent to the oligosaccharide compoundof Formula (VII) described above.

The oligosaccharide compound 7 is subjected to quaternary ammonium saltconversion, and followed by reaction with a halogenated hydrocarbon inan organic solvent by a conventional method in the art to obtain anoligosaccharide compound in which R₆ is a substituted or unsubstitutedC1-C6 hydrocarbon group or a C7-C12 aryl group.

The acetyl group on D-GalNAc in the oligosaccharide compound 7 can beremoved by a hydrazinolysis method to obtain a deacetylatedoligosaccharide compound. The deacetylated oligosaccharide compound canin turn be reacted with an acid anhydride or Et₃N.SO₃ to obtain anN-reacylated or resulfated oligosaccharide compound, namely, anoligosaccharide compound of Formula (VI) or Formula (VII) in which R₇ isa C2-C5 acyl or —SO₃H.

The reducing terminal -D-GlcA of the oligosaccharide compound 7 can beoptionally reacted with an alcohol compound under acidic conditions toform a terminal alkylation product, thereby obtaining the compound ofFormula (VI) in which R₁₀ is optionally a substituted or unsubstitutedC1-C6 hydrocarbon group or C7-C12 aryl group.

The aldehyde group at the C1 position of the -D-GlcA at the reducingterminal of the oligosaccharide compound 7 can be reductively aminatedin the presence of an organic amine. The reaction comprises reacting anorganic amine with the aldehyde group at the C1 position of the terminalglycosyl group to form a Schiff base, which is reduced to a secondaryamine in the presence of a reducing agent, thereby obtaining a compound(9) of Formula (VII) in which R₁₁ is —NHR₁₂.

The aldehyde group at the C1 position of the -D-GlcA at the reducingterminal of the oligosaccharide compound 7 may be optionally reduced toan alditol -D-GlcA-ol using a reducing agent such as sodium borohydride,and the -D-GlcA-ol may further optionally be reacted with an alcoholcompound under acidic conditions to form a terminal alkylation product,thereby obtaining the compound of Formula (VII) (9) in which R₁₁ is—OR₁₃, and R₁₃ is optionally —H, a substituted or unsubstituted C1-C6hydrocarbon group or a C7-C12 aryl group.

Obviously, the oligosaccharide compound represented by Formula (VI) orFormula (VII) having various specific structures defined by the presentinvention may be obtained by using the above structural modificationmethod in combination.

For the preparation method shown in Scheme 2, a preferred embodiment is:

in the step (a), the FG quaternary ammonium salt is benzethonium salt;the organic solvent is DMF or a DMF-ethanol mixture; the carboxylate isa benzyl ester, and “converting all or part to carboxylate” means thatthe degree of carboxyl esterification of Compound 3 is in the range offrom about 30% to about 100%

in the step (b), the organic solvent is DMF or a DMF-ethanol mixture,and the strong base is sodium ethoxide.

in the step (c), the conversion of the quaternary ammonium salt mixtureto the alkali metal salt means that a saturated aqueous solution ofsodium chloride is added to the reaction solution, thereby convertingthe obtained oligosaccharide homologues into a sodium salt form; thebasic hydrolysis in the aqueous solution means that the carboxylate ofthe homologous oligosaccharide compound is hydrolyzed in NaOH aqueoussolution with a concentration of 0.05 M to 1 M.

in the step (d), the chromatography includes, but is not limited to, gelchromatography and/or ion exchange chromatography.

in the step (e), the further structural modification includes, but isnot limited to, carboxyl esterification of D-GlcA and ΔUA in theoligosaccharide compound; deacetylation and optionally reacylation orresulfation of D-GalNAc; alkylation, reduction, reductive amination orreductive alkylation of the hemiacetal at the C1 position of thereducing terminal -D-GlcA.

Similarly, in the preparation method of the oligosaccharide compounddescribed in Scheme 2, the sulfated form of the obtained oligosaccharidecompound also depends on the sulfated form of the natural FG.

Therefore, natural FG derived from echinoderma such as Stichopusvariegatus, Stichopus horrens, and Stichopus monotuberculatus issuitable for the preparation of the oligosaccharide compound of Formula(X) and Formula (XI) and a pharmaceutically acceptable salt thereof, andthe compound is substantially an oligosaccharide compound of Formula(VI) and Formula (VII) in which R₁═R₃═R₄═R₅═—SO₃H, and R₂═—H.

In Formula (X) and Formula (XI), R₆, R₇, R₁₀ and R₁₁ are defined asabove.

However, natural FG from echinoderms such as Holothuria scabra,Holothuria fuscopunctata and Pearsonotheia graeffei is suitable for thepreparation of the oligosaccharide compound of Formula (XII) and Formula(XIII), and the compound is substantially an oligosaccharide compound ofFormula (VI) and Formula (VII) in which R₁═—H, and R₂═R₃═R₄═R₅═—SO₃ ⁻.

In Formula (XII) and Formula (XIII), R₆, R₇, R₁₀ and R₁₁ are defined asabove.

Obviously, the β-elimination reaction of natural FG under the technicalconditions of the present invention can also be used to prepare amixture of FG oligosaccharide compounds with more regular chemicalstructure.

Therefore, the present invention further provides a method for thepreparation of the oligosaccharide mixture of the present invention anda pharmaceutically acceptable salt thereof. Wherein, (1) the mixture iscomposed of a homologue of the oligosaccharide compound having thestructure represented by Formula (I) defined in the specification, andin the homologous oligosaccharide compounds of the structure of Formula(I), R₈ is the group simultaneously represented by Formula (II),simultaneously represented by Formula (III) or simultaneouslyrepresented by Formula (IV). Specifically, in the molar ratio, the ratioof the oligosaccharide compound of Formula (I) in which R₈ is the groupsimultaneously represented by Formula (II), or simultaneouslyrepresented by Formula (III) or simultaneously represented by Formula(IV) accounts for no less than 95% in the mixture. In the preparationmethod, natural FG is used as the starting material, and optionally, FGcarboxylate is subjected to β-elimination depolymerization and terminal“reduction reaction” in the presence of a strong base and a reducingagent to obtain a mixture of homologous oligosaccharide compounds; or FGcarboxylate is subjected to “β-elimination depolymerization” andterminal “peeling reaction” in the presence of a strong base to obtain amixture of homologous oligosaccharide compounds. Then, theoligosaccharide mixture with the desired molecular weight distributionis obtained by post-treatment and optional further substituent structuremodifications.

As described above, by treating natural product FG according to thesteps (a) to (c) shown in Scheme 1, a homologous oligosaccharide mixturehaving L-FucS-(α1-3)-ΔUA-1- at the non-reducing terminal and -D-GalNAcSat the reducing terminal can be obtained. Similarly, in the preparationmethod of the oligosaccharide mixture of the present invention, one ofthe methods is that: FG carboxylate is subjected to β-eliminationdepolymerization and terminal “reduction reaction” in the presence of astrong base and a reducing agent to obtain a mixture of homologousoligosaccharide compounds; the homologous oligosaccharide compoundcontained in the obtained oligosaccharide mixture has the generalstructure represented by Formula (I) defined above, and wherein R₈ is agroup represented by Formula (II) defined above. The method comprisesthe specific steps of:

(a) converting the natural FG into a quaternary ammonium salt form, andin an organic solvent, completely or partially converting the carboxylgroup on hexuronic acid residue in the obtained FG into a carboxylate;

(b) in an organic solvent, subjecting the FG carboxylate toβ-elimination depolymerization and terminal reduction reaction in thepresence of a reducing agent and a strong base, thereby obtaining amixture of homologous oligosaccharide compound having -D-GalNAc-ol atthe reducing terminal;

(c) converting the oligosaccharide mixture obtained in the step (b) intoan alkali metal salt, and subjecting the carboxylate of the homologousoligosaccharide compound to basic hydrolysis in an aqueous solution, toobtain a mixture of the homologous oligosaccharide compound containing afree carboxyl group, and performing appropriate post-treatment;

(d) optionally subjecting the oligosaccharide mixture obtained in thestep (d) to a further substituent structural modification.

In a preferred embodiment:

In the step (a), the quaternary ammonium salt is benzethonium salt; theorganic solvent is DMF or a DMF-ethanol mixture; the carboxylate is abenzyl ester; and the “complete or partial conversion into carboxylate”means that the degree of carboxyl esterification in FG is in the rangefrom about 30% to about 100%.

In the step (b), the organic solvent is DMF or a DMF-ethanol mixture;the reducing agent is sodium borohydride; and the strong base is sodiumethoxide.

In the step (c), the conversion of the quaternary ammonium salt mixtureto the alkali metal salt comprises adding a saturated aqueous solutionof sodium chloride to the reaction solution to convert the obtainedoligosaccharide homologue into a sodium salt form; the basic hydrolysisin the aqueous solution comprises hydrolyzing the carboxylate ofoligosaccharide compounds in NaOH aqueous solution with a concentrationof 0.05 M˜1 M.

As described above, the natural FG prepared according to the methods inthe prior art also typically contains some amount of fucan, glycogen,and hexosamine-containing polysaccharide, which have a molecular weightdistribution similar to FG These polysaccharide compositions have asmall change in molecular weight after being treated by the above steps(a) and (b). Therefore, in the post-treatment step described in the step(c), these polysaccharide impurities can be easily removed byultrafiltration method, dialysis method or gel chromatography.

As shown in the above Scheme 1, the depolymerized product obtained byβ-elimination depolymerization and terminal reduction treatment may alsohave a broader molecular weight distribution (in the oligosaccharidemixture 6 shown in Scheme 1, n may be an integer of about 0-15).Therefore, in the post-treatment step of the step (c), ultrafiltrationmethod, dialysis method or gel chromatography treatment may be selectedto remove the oligosaccharide with a higher degree of polymerization andthe small molecule compounds, thereby obtaining an oligosaccharidemixture with desired molecular weight distribution.

In the step (d), the further substituent structural modificationincludes, but is not limited to, carboxyl esterification of D-GlcA andunsaturated ΔUA in the oligosaccharide compounds; deacetylation andoptional further reacylation or resulfation of D-GalNAc;hydroxyalkylation at the C1 position of the reducing terminalD-GalNAc-ol.

Obviously, compared with the oligosaccharide mixture described in theinvention patent ZL 201310099800, the homologous oligosaccharide mixturein which R₈ is a group of Formula (II) according to the presentinvention has a more regular chemical structure. In the oligosaccharidecompound contained in the former, about 10% to 30% of theoligosaccharide compounds have D-GlcA (or a derivative thereof) at thereducing terminal, and the remaining oligosaccharide compounds haveD-GalNAc (or a derivative thereof) at the reducing terminal, however,the oligosaccharide compounds contained in the oligosaccharide mixtureof the present invention have D-GalNAc (or a derivative thereof) at thereducing terminal, and there is no or only a trace amount ofoligosaccharide compound having D-GlcA (or a derivative thereof) at thereducing terminal.

Further, as can be seen from the above, by treating the natural productFG according to the steps (a)˜(c) shown in Scheme 2, a homologousoligosaccharide mixture having L-FucS-(α1-3)-ΔUA-1- at the non-reducingterminal and the -D-GlcA at the reducing terminal can be obtained.Therefore, in the preparation method of the oligosaccharide mixture ofthe present invention, another method comprises: subjecting the FGcarboxylate to “β-elimination depolymerization” and terminal “peelingreaction” in the presence of a strong base to obtain a mixture ofhomologous oligosaccharide compounds; the homologous oligosaccharidecompounds contained in the obtained oligosaccharide mixture have ageneral structure represented by Formula (I) as defined in thespecification of the present invention, and R₈ is a group represented byFormula (III) or Formula (IV) defined above. The method comprises thespecific steps of:

(a) converting the natural FG into a quaternary ammonium salt form, andin an organic solvent, converting all or part of the carboxyl group onthe hexuronic acid residue in the obtained FG into a carboxylate;

(b) in an anhydrous organic solvent, treating the FG carboxylate with astrong base to cause β-elimination depolymerization, followed by addinga small amount of aqueous solution of a strong base, subjecting the FGdepolymerized product to further “peeling reaction” to lose the-D-GalNAc residue at the reducing terminal, thereby obtaining a mixtureof homologous oligosaccharide compounds having -D-GlcA at the reducingterminal.

(c) converting the oligosaccharide mixture obtained in the step (b) intoan alkali metal salt, and subjecting the carboxylate of the homologousoligosaccharide mixture in an aqueous solution to basic hydrolysis, toobtain a mixture of the homologous oligosaccharide compounds containinga free carboxyl group, and performing appropriate post-treatment;

(d) optionally subjecting the oligosaccharide mixture obtained in thestep (c) to a further substituent structural modification.

In a preferred embodiment:

In the step (a), the quaternary ammonium salt is a benzethonium salt;the organic solvent is DMF or a DMF-ethanol mixture; the carboxylate isa benzyl ester; and the degree of carboxyl esterification of the FGcarboxylate is in the range from about 30% to about 100%.

In the step (b), the organic solvent is DMF or a DMF-ethanol mixture;and the strong base is sodium ethoxide; the small amount of aqueoussolution of a strong base refers to a 1 M˜2 M NaOH aqueous solution thatis equivalent to about ⅕ to 1/10 of the total volume of the reactionsolution.

In the step (c), the conversion of the quaternary ammonium salt mixtureto the alkali metal salt comprises adding a saturated aqueous solutionof sodium chloride to the reaction solution to convert the obtainedoligosaccharide homologue into a sodium salt form; the basic hydrolysisin the aqueous solution means that the carboxylate of theoligosaccharide compounds is hydrolyzed in NaOH aqueous solution with aconcentration of 0.05 M˜1 M. Similarly, in the post-treatment, gelchromatography, ultrafiltration and/or dialysis may be optionally usedto remove the undepolymerized macromolecular polysaccharide impuritiesand remove the highly polymerized oligosaccharide compounds and smallmolecular impurities, and thus obtain an oligosaccharide mixture of thedesired molecular weight range.

In the step (d), the further substituent structural modificationincludes, but is not limited to, carboxyl esterification of D-GlcA andΔUA in the oligosaccharide compounds; deacetylation and optional furtherreacylation or resulfation of D-GalNAc; alkylation, reduction, reductiveamination or reductive alkylation of the hemiacetal at the C1 positionof the reducing terminal -D-GlcA.

Similarly, compared with the oligosaccharide mixture described in theinvention patent ZL 201310099800, the homologous oligosaccharide mixturein which R₈ is a group of Formula (III) or Formula (IV) according to thepresent invention has a more regular chemical structure. In theoligosaccharide compounds contained in the former, about 10% to 30% ofthe oligosaccharide compounds have D-GlcA (or a derivative thereof) atthe reducing terminal, and the remaining oligosaccharide compounds haveD-GalNAc (or a derivative thereof) at the reducing terminal; however,the oligosaccharide compounds contained in the oligosaccharide mixtureof the present invention have D-GlcA (or a derivative thereof) at thereducing terminal, and have no or only a trace amount of oligosaccharidecompounds having D-GalNAc (or a derivative thereof) at the reducingterminal.

Obviously, using the natural FG derived from an echinoderma such as S.variegatus, S. horrens and S. Monotuberculatus as a starting material,according to the preparation method of the oligosaccharide mixturedescribed in present invention, the mixture of homologousoligosaccharide compounds of Formula (VIII), Formula (X) and Formula(XI) described above can be prepared. Using the natural FG derived fromechinoderma such as H. scabra, H. fuscopunctata and P. graeffei as astarting material, according to the preparation method of theoligosaccharide mixture described in present invention, the mixture ofhomologous oligosaccharide compounds of Formula (IX), Formula (XII) andFormula (XIII) described above can be prepared.

The available data show that in a homologous oligosaccharide compoundobtained by deacylated-deaminated depolymerization of the natural FGcontaining a L-Fuc_(2S4S) side chain substituent, nonasaccharide (NSac)is the smallest structural fragment with potent inhibitory activity offactor Xase (Zhao L Y et al., PNAS, 2015, 112: 8284-8289.). The presentinventors have conducted a structure-activity relationship study on theactivity of the intrinsic factor Xase (factor Xase derived from humanand experimental animals) of the purified oligosaccharide of the presentinvention and found that:

(1) The oligosaccharide compounds of the present invention have aselective activity of inhibiting intrinsic factor Xase. In general,using an in vitro enzyme activity assay, the IC₅₀ value of theoligosaccharide compounds inhibiting Factor Xase of the presentinvention may be in the range of about 5 to 200 ng/ml. The selectiveinhibition of the activity of the intrinsic factor Xase means that inthe presence or absence of antithrombin (AT), these oligosaccharidecompounds, at a concentration of significantly inhibiting Xase, have nosignificant effect on the activity of coagulation factors and platelets,but may have a certain intensity of heparin cofactor II(HC-II)-dependent IIa inhibitory activity.

(2) For a series of oligosaccharide compounds containing 3mmonosaccharide groups and having ΔUA at the non-reducing terminal and-D-GalNAc-ol at the reducing terminal, the minimum structural fragmentthat potently inhibits Xase activity is also nonasaccharide (NSac). Theresults show that the glycosyl structure changes at the non-reducingterminal and the reducing terminal has little effect on the inhibitoryactivity of factor Xase.

(3) For a series of oligosaccharide compounds containing (3m-1)monosaccharide residues and having -D-GlcA(-ol) at the reducingterminal, the minimum structural fragment for potent inhibitory activityof factor Xase is octasaccharide (OSac). The results suggest that thereducing terminal -D-GalNAc-ol in the above NSac may not be an essentialstructure for its potent inhibitory activity of factor Xase.

(4) In general, the oligosaccharide compounds of the present inventionhaving a degree of polymerization of not less than OSac have potentintrinsic factor Xase inhibitory activity (IC₅₀ values are less thanabout 100 ng/ml); the oligosaccharides having a higher degree ofpolymerization have a slightly enhanced activity.

(5) In the oligosaccharide compounds of the present invention,hexasaccharide or pentasaccharide at a higher concentration may have acertain inhibitory activity against intrinsic Xase, although theactivity intensity thereof is relatively weak; further, hexasaccharideand pentasaccharide also have a certain intensity of heparin cofactor II(HC-II)-dependent IIa inhibitory activity.

(6) In the oligosaccharide compounds of the present invention, thesubstituent structural modification can significantly affect thephysicochemical properties of the oligosaccharide compounds, such aswater solubility and oil-water partition coefficient, but generally havea small effect on its coagulation factor inhibitory activity andanticoagulant and antithrombotic activity.

(7) The oligosaccharide compounds of the present invention may havesignificant anticoagulant activity for inhibiting intrinsic coagulationpathway, based on prolonging the activated partial thromboplastin time(APTT) of human normal plasma, the concentration of the drug requiredfor multiplying APTT is generally in the range of about 2˜18 μg/mL. Andthe oligosaccharide compounds of the present invention have nosignificant effect on extrinsic coagulation.

(8) In the pathological model of the experimental animals, theoligosaccharide compounds of the present invention can significantlyinhibit arteriovenous thrombosis. For example, the inventors' researchesshow that in various experimental animal models, based on the weight ofthe thrombus, when a compound of the present invention (such asnonasaccharide or octasaccharide) is administered subcutaneously (sc) orintravenously (iv) at a dose of about 2 mg/kg˜20 mg/kg, the inhibitionrate on experimental venous thrombosis (for example, caused by inferiorvena cava ligation) may reach 70%˜100%. And at an equivalentantithrombotic dose, the effect of the oligosaccharide compound onbleeding time and bleeding volume may be significantly lower than thatof low molecular weight heparin drugs used clinically.

(9) The oligosaccharide mixture of the present invention has aninhibitory activity against intrinsic factor Xase and an anticoagulantand antithrombotic activity, which is similar to a purifiedoligosaccharide compound.

In summary, the oligosaccharide compounds of the present invention andmixtures thereof have significant anticoagulant and antithromboticactivity, and when the degree of oligosaccharide polymerization is notlower than that of octasaccharide, both the oligosaccharide compounds ofthe present invention and the mixture thereof are an intrinsic factorXase inhibitor with good selectivity. Existing research data show thatintrinsic coagulation pathway is closely related to pathologicalthrombosis, and may not be necessary for physiological hemostasis.Selective intrinsic coagulation pathway inhibitors may inhibitpathological thrombosis, and bleeding tendency may be effectivelyreduced. Since factor Xase is the terminal and rate-limiting enzymeactive site of the intrinsic coagulation pathway, intrinsic factor Xasehas become a drug target for the development of anticoagulant andantithrombotic drugs with low bleeding tendency.

In view of the significant anticoagulant and antithrombotic activity ofthe oligosaccharide and the oligosaccharide mixture of the presentinvention, these oligosaccharide and oligosaccharide mixture should haveclinical application value of prevention and/or treatment of thromboticdiseases. Therefore, the present invention further provides apharmaceutical composition comprising the oligosaccharide or theoligosaccharide mixture.

First, the present invention provides a pharmaceutical compositionhaving antithrombotic activity. The pharmaceutical composition comprisesan effective antithrombotic dose of the oligosaccharide compound of thepresent invention or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable excipient. Wherein, the oligosaccharidecompound refers to a compound having the structure represented byFormula (I) as defined in the present invention.

In view of the physicochemical properties of the oligosaccharidecompounds of the present invention, the pharmaceutical composition ofthe present invention is preferably prepared into a parenteral dosageform, such as an aqueous solution for injection or a lyophilizedpreparation formulated as an aqueous solution for injection before use,and may also be a spray administered by the respiratory tract, or atransdermal patch, a paste or a gel for transdermal administration, andso on.

The oligosaccharide compounds of the present invention generally havegood water solubility and are easily formulated into aqueous solutions;since the active ingredients have low molecular weights, pathogenicmicroorganisms and pyrogens may be removed by ultrafiltration; theoptional pharmaceutical excipients for the aqueous solution and/orlyophilized preparation may include inorganic salts such as sodiumchloride, buffer salts for adjusting the osmotic pressure and/or pH ofthe solution, and preferably include no co-solvent and/or surfactant.For the lyophilized powder formulated into liquid injection before use,besides the inorganic salt and/or buffer salt, a pharmaceuticallyacceptable excipient which facilitates formulation of the preparationsuch as mannose may be selected.

In general, the oral bioavailability of the oligosaccharide compounds isrelatively limited, but the oligosaccharide compounds of the presentinvention (especially the oligosaccharides obtained by substituentstructural modifications) may still have certain pharmacodynamicactivity when administered by the gastrointestinal tract. Thus, thepharmaceutical compositions of the present invention may also beformulated into gastrointestinal dosage forms well known to thoseskilled in the art, such as a tablet, a capsule.

Those skilled in the art will appreciate that for the pharmaceuticalcomposition in a particular formulation form, the effectiveantithrombotic dose of the oligosaccharide compound and itspharmaceutically acceptable salt is related to the factors such as thedosage form, the route of administration, and the weight andphysiological state of the patient. In general, in the unit preparationform of the pharmaceutical composition of the present invention, thecontent of the oligosaccharide active ingredient is in the range ofabout 5 mg˜100 mg; in the unit preparation form of the preferredpharmaceutical composition, the content of the oligosaccharide as anactive ingredient may be in the range of about 20 mg˜80 mg.

Similarly, the present invention also provides a pharmaceuticalcomposition having antithrombotic activity. The pharmaceuticalcomposition comprises a potent antithrombotic dose of theoligosaccharide mixture of the present invention or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient.

For the pharmaceutical composition comprising the oligosaccharidemixture of the present invention, the preparation form and dosageselection associated with the administration route are similar to thatof the oligosaccharide-containing pharmaceutical composition mentionedabove. For example, a preferred administration route is parenteraladministration, especially subcutaneous injection administration orintravenous injection administration; a preferred preparation form isaqueous solution for injection or a lyophilized powder for injection; ina unit dosage form of a preferred pharmaceutical composition, theoligosaccharide as an active ingredient may be present in an amountranging from about 20˜100 mg.

The oligosaccharide compound, the oligosaccharide mixture and thepharmaceutically acceptable salt thereof of the present invention havepotent anticoagulant and antithrombotic activity and may be used for theprevention and treatment of thrombotic diseases, such as thromboticcardiovascular diseases, thrombotic cerebrovascular disease, pulmonaryvein thrombosis, peripheral venous thrombosis, deep vein thrombosis,peripheral arterial thrombosis. Therefore, the present invention alsoprovides the use of the oligosaccharide compound and/or oligosaccharidemixture and a pharmaceutically acceptable salt thereof in thepreparation of a medicament for the treatment and/or prevention ofthrombotic diseases. The thrombotic diseases include, but are notlimited to, venous thrombosis, arterial thrombosis and/or ischemiccardiovascular and cerebrovascular diseases.

Similarly, the present invention further provides the use of thepharmaceutical composition comprising the oligosaccharide compoundand/or oligosaccharide mixture and a pharmaceutically acceptable saltthereof for the preparation of a medicament for treating and/orpreventing thrombotic diseases. The thrombotic diseases include, but arenot limited to, venous thrombosis, arterial thrombosis, and/or ischemiccardiovascular and cerebrovascular diseases.

Abbreviations

FG Fucosylated glycosaminoglycan Fucosylated glycosaminoglycan L-Fuc (F)L-fucose L-fucose L-FucS Sulfated fucose L-fucose sulfate L-Fuc2S4SL-2,4-bis-O-sulfo-fucose L-fucose-2,4-disulfate L-Fuc3S4SL-3,4-bis-O-sulfo-fucose L-fucose-3,4-disulfate D-GalNAc (A)β-D-N-acetyl-2-deoxy-2-amino-galactoseD-N-acetyl-2-deoxy-2-amino-galactose (D-N-acetylgalactosamine)(D-acetylgalactosamine) D-GalNAc-olβ-D-N-acetyl-2-deoxy-2-amino-galactitolD-N-acetyl-2-deoxy-2-amino-galactitol D-GalNAcS D-GalNAc sulfateD-acetylgalactosamine sulfate D-Ga1NAc4S6SD-acetylgalactosamine-4,6-disulfate D-acetylgalactosamine-4,6-disulfateD-GlcA (U) D-glucuronic acid D-glucuronic acid ΔUA (ΔU)L-4-deoxy-threo-hex-4-enopyranosyluronic acid 4-deoxy-L-threo-hex-4-(4,5-unsaturated hexenuronic acid) enopyranosyluronic acid (unsaturatedhexenuronic acid) Xase tenase complex Factor X enzyme complex HC-IIheparin cofactor II heparin cofactor II APTT activated partialthromboplastin time activated partial thromboplastin time PT prothrombintime prothrombin time TT thrombin time thrombin time NSac nonasaccharidenonasaccharide OSac octasaccharide octasaccharide DMSO dimethylsulfoxide dimethyl sulfoxide DMF N,N-DimethylformamideN,N-Dimethylformamide

DESCRIPTION OF THE DRAWINGS

FIG. 1. HPGPC profiles of Compounds A1˜A4

FIG. 2. ¹H NMR spectrum and assignments for Compound A1

FIG. 3. ¹³C NMR spectrum and assignments for Compound A2

FIG. 4. ¹³C-¹H HSQC spectrum and assignments for Compound A3

FIG. 5. Q-TOF MS spectrum and assignments for Compound A1

FIG. 6. ¹H NMR spectrum and assignments for Compound B1

FIG. 7. ¹³C NMR spectrum and assignment for Compound B2

FIG. 8. ¹³C-¹H HSQC spectrum and assignments for Compound B3

FIG. 9. Q-TOF MS spectrum and assignments for Compound B2

FIG. 10. HPGPC profiles of Compound B6-B8

FIG. 11. ¹³C NMR spectrum and assignments for Mixture C1

FIG. 12. HPLC profile of Mixture D1

FIG. 13. ¹³C NMR spectrum and assignments for Mixture D1

FIG. 14. Effect of A2 and D1 on thrombosis of the inferior vena cava in

FIG. 15. Effect of A2 and D1 on the amount of bleeding loss in mice

DETAILED DESCRIPTION OF THE INVENTION

The following examples are intended to describe the contents of thepresent invention in detail, but do not limit the scope of the presentinvention.

Example 1

Preparation of Compounds A1, A2, A3, A4 and A5:

L-2,4-disulfated fucosyl-(α1→3)-L-4-deoxy-threo-hex-4-enepyranosyluronicacid-(α1→3)-{-D-N-acetyl-2-deoxy-2-amino-4,6-disulfatedgalactosyl-(β1→4)-[L-2,4-disulfatedfucosyl-(α1→3)]-D-glucuronyl-(β1→3)}_((n+1))-D-N-acetyl-2-deoxy-2-amino-4,6-disulfatedgalactitol (n=0, 1, 2, 3 and 4; hexasaccharide, nonasaccharide,dodecasaccharide, pentadecasaccharide and octadecasaccharide)

1.1 Materials

SvFG, Natural FG (sodium salt) from Stichopus variegatus, which wasprepared according to the literature method (Zhao L Y et al., PNAS,2015, 112: 8284-8289), with a purity of 98% (HPGPC, area normalizationmethod) and a weight average molecular weight (Mw) of about 70 kDa.

The reagents used such as benzethonium chloride, benzyl chloride, DMF,sodium hydroxide, sodium chloride, and ethanol were all commerciallyavailable analytical reagents. Sephadex G10, medium (50-100 m), GEHealthcare; Bio-Gel P-6/P-2 gel, fine (45-90 m), Bio-Rad; Bio-Gel P-10gel, medium (90-180 m), Bio-Rad; HPLC Chromatograph, Agilent 1200/1260Series Chromatograph.

1.2 Methods

(1) Quaternary ammonium salt conversion of SvFG: 2.0 g of SvFG wasdissolved in 30 mL of deionized water; and 5.0 g of benzethoniumchloride was dissolved in another 80 mL of deionized water. The SvFGsolution was titrated with the benzethonium chloride solution withstirring to give a white precipitate. The obtained precipitate waswashed three times with 55 mL of deionized water and dried under vacuumto give 5.34 g of SvFG quaternary ammonium salt.

(2) Carboxyl esterification of SvFG: The SvFG quaternary ammonium saltobtained in the step (1) was placed in a round bottom flask, dissolvedin 26 mL of DMF, then added with 0.769 mL of benzyl chloride, reacted at35° C. for 24 h with stirring; and allowed to stand and let the solutioncool to room temperature (25° C.). The product sample was taken for ¹HNMR detection and the degree of carboxyl esterification of the FG wascalculated to be about 41%.

(3) β-elimination depolymerization in the presence of a reducing agent:a freshly prepared 8.9 mL of 0.08 M sodium ethoxide-ethanol solution(containing 0.4 M NaBH₄) was added to the reaction solution of the step(2), and stirred for 30 min.

(4) Sodium salt conversion and carboxyl ester hydrolysis of thedepolymerized product: 35 mL of a saturated NaCl solution and 284 mL ofabsolute ethanol were added to the reaction solution of the step (3),centrifuged at 4000 rpm×10 min to obtain a precipitate. The obtainedprecipitate was dissolved in 90 mL of water, added with 1.5 mL of 6 MNaOH solution, and stirred at room temperature for 30 min, and thendropwise added with 6 M HCl to neutralize the reaction solution (pH˜7.0). The reaction solution was filtered through a 0.45 μm filter, andthe obtained filtrate was desalted by a G10 gel column chromatographyand lyophilized to obtain a total of 1.059 g of depolymerized productdSvFG (depolymerized SvFG) (yield 53%).

(5) Isolation and purification of Compounds A1˜A5: 1 g of dSvFG wasdissolved in 10 mL of 0.2 M NaCl, loaded on a Bio-Gel P-10 gel column(Ø2 cm, 1 200 cm), eluted with 0.2 M NaCl solution at a flow rate 10mL/h, and the eluate fractions of 2.5 mL/tube were collected. The eluatefractions were monitored and the elution profiles were plotted by thecysteine-sulfuric acid method, and the eluate fractions having the samecompositions were combined. Purity was determined by HPGPC method (TSKgel G2000SW XL, Ø 7.8 mm×l 300 mm column). Unpurified samples werefurther purified on a Bio-Gel P-10 gel column. Purified oligosaccharideswere desalted on a Sephadex G-10 or Bio-Gel P-2 gel column and thenlyophilized.

(6) Spectral analysis: ¹H-/¹³C- and 2D-NMR were detected using BrukerDRX 800 MHz NMR spectrometer with a spectral width of 16025.6 Hz, anacquisition time of 2.0447 s, a pulse width of 9.5 s, a relaxation timeof 1 s, and a scan of 32 times. The sample had a concentration of(10-15) g/L, and was repeatedly lyophilized three times with heavy waterbefore the test; ESI-Q-TOF MS was analyzed by micrOTOF-QII ESI-MS(Bruker, Germany) mass spectrometer. The mass spectrometry conditionswere: capillary voltage 2500 V, nebulizer voltage 0.6 bar, dry gas flowrate 4.0 L/min, dry gas temperature+180° C., m/z scan range 50˜3000.Data were analyzed using Bruker Compass Data-Analysis 4.0(Bruker-Daltonics, Germany) software.

1.3 Results

(1) Compound A1 35 mg, A2 45 mg, A3 55 mg, A4 35 mg, and A5 20 mg wereobtained by the method described, and the purity was determined to beabout 99% by HPGPC method. The HPGPC patterns of oligosaccharidecompounds A1˜A5 are shown in FIG. 1.

(2) Structural analysis of Compounds A1˜A5: The ¹H NMR spectrum andassignments for oligosaccharide compound A1 are shown in FIG. 2; the ¹³CNMR spectrum and assignments for Compound A2 are shown in FIG. 3; the¹³C-¹H HSQC spectrum and assignments for Compound A3 are shown in FIG.4; the Q-TOF MS spectrum and assignments for Compound A1 are shown inFIG. 5; the ¹H/¹³C NMR signal assignments for Compounds A1˜A2 are shownin Tables 1 and 2, respectively.

According to ¹H-/¹³C-, 2D-NMR and Q-TOF MS analysis, the chemicalstructure of Compounds A1˜A5 isL-Fuc_(2S4S)-(α1,3)-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-GlcA-(β1,3)}_(n+1)-D-GalNAc_(4S6S)-ol,wherein n=1, 2, 3, 4 and 5, that is, Compounds A1˜A5 are hexasaccharide,nonasaccharide, dodecasaccharide, pentadecasaccharide andoctadecasaccharide, respectively, having the chemical structural formulaof:

In A1, n=1, in A2, n=2; in A3, n=3; in A4, n=4; in A5, n=5.

TABLE 1 ¹H/¹³C NMR signal assignments and coupling constant of CompoundA1 (ppm, Hz) rA U F A dU dF H-1 3.636 4.536 5.615 4.550 4.838 5.432J_(1, 2) = 6.72 J_(1, 2) = 8.22 J_(1, 2) = 3.72 J_(1, 2) = 8.28 J_(1, 2)= 7.98 J_(1, 2) = 3.60 H-2 4.199 3.666 4.426 4.079 3.820 4.355 J_(2, 3)= 7.80 J_(2, 3) = 8.61 J_(2, 3) = 9.72 J_(2, 3) = 9.60 J_(2, 3) = 8.34J_(2, 3) = 10.44 H-3 4.220 3.721 4.095 4.088 4.438 4.055 — J_(3, 4) =8.94 J_(3, 4) = 4.02 — J_(3, 4) = 2.46 J_(3, 4) = 2.64 H-4 4.328 3.8794.795 4.912 5.688 4.623 — J_(4, 5) = 9.18 — — / — H-5 4.343 3.713 4.8923.989 4.282 — J_(5, 6) = 6.36 J_(5, 6,6′) = 7.08, 4.62 J_(5, 6) = 6.78H-6 4.090 1.320 4.245/4.145 1.231 — J_(6, 6′) = 11.10 Ac—CH₃ 1.947 1.985C-1 62.64 106.03 99.70 102.73 106.31 99.19 C-2 54.57 77.29 77.96 54.3473.28 77.90 C-3 78.08 80.35 69.47 79.10 79.33 69.37 C-4 80.80 78.3484.08 79.22 109.30 83.63 C-5 70.48 79.22 69.23 74.80 149.97 69.25 C-672.63 177.63 18.78 70.42 171.83 18.59 (Ac) C═O 177.19 177.97 (Ac) CH₃25.02 25.46 Note: rA represents D-GalNAc-ol at the reducing terminal; dUand dF represent ΔUA and L-Fuc linked to ΔUA.

TABLE 2 ¹H/¹³C NMR signals assignments for Compound A2 (ppm, Hz) rA rUrF A U F dA dU dF H-1 3.626 4.520 5.615 4.512 4.395 5.628 4.530 4.8325.435 H-2 4.189 3.661 4.412 4.010 3.579 4.412 4.047 3.830 4.348 H-34.211 3.737 4.093 3.956 3.688 4.093 4.073 4.446 4.060 H-4 4.403 3.9584.803 4.711 3.880 4.803 4.908 5.686 4.623 H-5 4.327 3.706 4.863 3.9193.600 4.863 3.971 4.288 H-6 4.079 1.299 4.206/4.123 1.311 4.230/4.1221.231 Ac—CH₃ 1.947 1.980 1.988 C-1 62.62 105.92 99.58 102.71 106.9299.38 102.68 106.02 99.10 C-2 54.53 77.23 77.89 54.19 76.76 77.90 54.2073.22 77.84 C-3 78.06 80.15 69.41 78.14 79.93 69.41 78.88 79.23 69.32C-4 80.76 78.33 83.96 79.18 78.33 83.96 79.09 109.26 83.54 C-5 70.4379.10 69.24 74.69 79.69 69.20 74.65 149.95 69.20 C-6 72.59 177.62 18.8670.27 177.88 18.86 70.21 171.78 18.59 (Ac) C═O 177.15 177.88 177.94 (Ac)CH₃ 24.99 25.52 25.45 Note: in the table, rA, rU and rF representGalNAc-ol at the reducing terminal, D-GlcA and L-Fuc glycosyl near thereducing terminal, respectively; dU, dA and dF represent AUA, D-GalNAclinked to AUA, and L-Fuc linked to AUA, respectively.

[Example 2] Preparation of Compounds B1, B2, B3, B4 and B5

L-3,4-disulfatedfucosyl-(α1,3)-L-4-deoxy-threo-hex-4-enepyranuronyl-(α1,3)-{D-N-acetyl-2-deoxy-2-amino-4,6-disulfated galactosyl-(β1,4)-[L-3, 4-disulfatedfucosyl-(α1,3)-]D-glucuronyl-(β1,3)}_(n)-D-N-acetyl-2-deoxy-2-amino-4,6-disulfatedgalactose-[L-3,4-disulfated fucosyl-(α1,3)-]-L-gulonic acid (n=0, 1, 2,3 and 4, pentasaccharide, octasaccharide, hendecasaccharide,tetradecasaccharide, and heptadecasaccharide).

2.1 Materials

HsFG Natural FG (sodium salt) from Holothuria fuscopunctata; which wasprepared according to the literature method (Zhao L Y et al., PNAS,2015, 112: 8284-8289), with a purity of 98% (HPGPC method), and a weightaverage molecular weight (Mw) of about 50 kDa.

The used reagents such as benzethonium chloride, benzyl chloride, DMF,sodium hydroxide, sodium chloride, and ethanol were all commerciallyavailable analytical reagents.

Sephadex G10/G25, medium (50-100 μm), GE Healthcare; Bio-Gel P-2 gel,fine (45-90 μm), Bio-Rad; Bio-Gel P-10 gel, medium (90-180 μm), Bio-Rad;1200/1260 Series HPLC Chromatograph, Agilent.

2.2 Methods

(1) Quaternary ammonium salt conversion of HsFG: 3.5 g of HsFG wastreated according to the method described in 1.2 (1) of Example 1,obtaining 10.3 g of HsFG quaternary ammonium salt.

(2) Carboxyl esterification of HsFG:HsFG quaternary ammonium salt wastreated according to the method described in 1.2 (2) of Example 1 toobtain HsFG carboxylate, and the sample was taken for ¹H NMR detection,and the degree of carboxyl esterification of the obtained product wascalculated to be about 44%;

(3) β-elimination depolymerization and terminal peeling reaction ofHsFG: To the reaction solution obtained in the step (2), a freshlyprepared 16.7 mL of 0.08 M sodium ethoxide-ethanol solution was added,and stirred at room temperature for 30 min, and then 2.5 mL of 2 M NaOHsolution was added, and stirred at 60° C. for 30 min.

(4) Sodium salt conversion, carboxylic ester hydrolysis and terminalreduction of depolymerized product: To the reaction solution in the step(3) was added sequentially 67 mL of saturated NaCl solution, 536 mL ofabsolute ethanol, centrifuged at 4000 rpm for 10 min; the obtainedprecipitate was dissolved in 120 mL of water, added with 1.0 mL of 6 MNaOH solution, and stirred at room temperature for 30 min; NaBH₄ wasadded to a final concentration of about 0.1 M, and stirred at roomtemperature for 30 min, then 6 M HCl was added dropwise to neutralizethe reaction solution (pH ˜7.0). The reaction solution was filteredthrough a 0.45 μm filter, and the filtrate was ultrafiltered through a30 kDa ultrafiltration membrane; the ultrafiltrate was concentrated anddesalted by G25 gel column chromatography and lyophilized to obtain 1.53g of depolymerized product dHsFG (yield 43.7%).

(5) Isolation and purification of Compounds B1˜B5: 1 g of dHsFG in thestep (4) was dissolved in 10 mL of 0.2 M NaCl, loaded on a Bio-Gel P-10gel column (Ø2 cm, 1 200 cm), eluted with 0.2 M NaCl solution at a flowrate 15 mL/h, and the eluate fractions of 2.5 mL/tube were collected. UVspectrophotometry (λmax 234 nm) was used for monitoring. HPGPC (TSKG2000 SW column) was used to detect the sample purity and composition ofthe eluate fractions. The unpurified fractions were continued to bepurified on a Bio-Gel P-10 gel column until the HPGPC spectrum of theproduct exhibited a single elution peak. The purified fractions weredesalted on a Sephadex G-10 or Bio-Gel P-2 column and then lyophilized.

(6) Spectral analysis: the same as the method described in 1.2 (6) ofExample 1, ¹H-/¹³C- and 2D-NMR was detected using Bruker DRX 800 MHz NMRspectrometer, ESI-Q-TOF MS was analyzed using microTOF-QII ESI-MS(Bruker, Germany) mass spectrometer. The detected data were analyzedusing Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany)software.

2.3 Results

(1) Compound B1 54 mg, B2 177 mg, B3 154 mg, B4 86 mg, B5 57 mg wereobtained by the method described above, and the purity was determined tobe >99% by HPGPC method.

(2) Structure analysis of Compounds B1˜B5: The ¹H NMR spectrum ofoligosaccharide Compound B1 is shown in FIG. 6; the ¹³C NMR spectrum andassignments for Compound B2 are shown in FIG. 7; the ¹³C-¹H HSQCspectrum and assignments of Compound B3 are shown in FIG. 8; the Q-TOFMS spectrum and assignments for Compound B2 are shown in FIG. 9; the¹H/¹³C NMR signal assignments for Compounds B1˜B2 are shown in Tables 3and 4, respectively.

Combined with ¹H-/¹³C-/2D-NMR and Q-TOF MS analysis, the chemicalstructure of Compounds B1 B5 isL-Fuc_(3S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(3S4S)-(α1,3)]-D-GlcA-(β1,3)}n-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(3S4S)-(α1,3)]-D-GlcA-ol(wherein n=0, 1, 2, 3 and 4). That is, Compounds B1˜B5 arepentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide,and heptadecasaccharide, respectively, having the chemical structuralformula of:

In B1, n=0; in B2, n=1; in B3, n=2; in B4, n=3; in B5, n=4.

TABLE 3 ¹H/¹³C NMR signal assignments and coupling constants forCompound B1 (ppm, Hz) rU rF rA dU dF H-1 3.743/3.700 5.039 4.639 4.8595.201 J_(1, 1') = 11.82 J_(1, 2) = 3.96 J_(1, 2) = 8.52 J_(1, 2) = 8.64J_(1, 2) = 3.78 H-2 4.060 3.886 4.043 3.822 3.886 J_(1/1', 2) =3.96/6.78 J_(2, 3) = 10.38 J_(2, 3) = 8.70 J_(2, 3) =7.56 J_(2, 3) =10.38 H-3 4.019 4.546 4.167 4.418 4.529 — J_(3, 4) = 2.76 J_(3, 4) =2.16 J_(3, 4) = 2.40 J_(3, 4) = 2.82 H-4 4.071 4.831 4.918 5.693 4.831J_(4, 5) = 7.44 — — — H-5 4.256 4.363 4.019 4.273 J_(5, 6) = 6.84J_(5, 6/6') = 8.24, 3.52 J_(5, 6) = 6.48 H-6 1.240 4.268/4.146 1.231J_(6, 6') = 10.68 (Ac)-CH₃ 1.986 C-1  65.28 104.26 104.31 105.82101.04/175.8 C-2  72.59  69.39  54.35  72.90 69.27 C-3  82.44  77.93 78.56  79.12 77.96 C-4  75.17  81.70  78.94 109.52 81.72 C-5  75.17 69.55  74.64 149.51 69.02 C-6 180.08  18.85  70.64 171.65 18.56 (Ac)-C= O 177.65 (Ac)-CH₃  25.19 Note: in the table, rU and dU representD-GlcA-ol at the reducing terminal and ΔUA at the non-reducing terminal,respectively; rF and dF represent L-Fuc glycosyl groups linked to thereducing terminal D-GlcA-ol and ΔUA, respectively. A representsD-GalNAc.

TABLE 4 ¹H-/¹³C-NMR signal assignments for Compound B2 (ppm, Hz) rU rFrA U F A dU dF H-1 3.733/3.691 5.033 4.622 4.426 5.286 4.530 4.846 5.204H-2 4.066 3.886 3.976 3.546 3.871 4.090 3.830 3.886 H-3 4.012 4.5453.981 3.632 4.458 4.087 4.426 4.530 H-4 4.012 4.837 4.755 3.954 4.9734.912 5.686 4.831 H-5 4.248 4.370 4.022 3.641 4.799 4.019 4.275 H-61.241 4.211/4.129 1.343 4.321/4.222 1.231 Ac—CH₃ 1.992 1.997 C-1 65.21104.17 104.17 106.4 101.97 102.39 105.8 100.9 C-2 72.59 69.24 54.2676.24 69.16 54.26 72.96 69.10 C-3 82.33 77.91 78.07 81.88 78.07 78.5579.10 77.96 C-4 75.03 81.68 78.87 77.91 82.09 78.98 109.4 81.69 C-574.62 69.52 75.03 79.86 69.00 74.62 149.6 69.36 C-6 180.08 18.84 70.52177.88 18.78 69.99 171.6 18.56 (Ac) —C═O 177.74 177.74 (Ac) —CH₃ 25.2825.26 Note: in the table, rA represent GalNAc near the reducingterminal, and rU and dU represent D-GlcA-ol at the reducing terminal andAUA at the non-reducing terminal, respectively; rF and dF representL-Fuc glycosyl linked to D-GlcA-ol at the reducing terminal and AUA,respectively.

[Example 3] Preparation of Compounds B6, B7, B8

L-3,4-disulfatedfucosyl-(α1,3)-L-4-deoxy-threo-hex-4-enopyanosyluronyl-(α1,3)-{D-N-acetyl-2-deoxy-2-amino-4,6-disulfatedgalactosyl-(β1,4)-[L-3,4-disulfatedfucosyl-(α1,3)-]D-glucuronyl-(β1,3)}_(n)-D-N-acetyl-2-deoxy-2-amino-4,6-disulfatedgalactosyl-(β1,4)-[L-3,4-disulfated fucosyl-(α1,3)-]-D-glucuronic acid(n=0, 1 and 2)

3.1 Materials:

HsFG, FG sodium salt derived from Holothuria fuscopunctata, derived fromthe same as described in 2.1 of Example 2.

The reagents used such as benzethonium chloride, benzyl chloride, DMF,sodium hydroxide, sodium chloride and ethanol were all commerciallyavailable analytical reagents.

3.2 Methods:

(1) Quaternary ammonium salt conversion of HsFG: 9.55 g of HsFGquaternary ammonium salt was prepared from 3.5 g of HsFG by the methodas described in 2.2(1) of Example 2.

(2) Carboxyl esterification of HsFG: carboxyl esterified HsFG wasobtained by the method described in 2.2 (2) of Example 2, and the degreeof carboxyl esterification was determined to be about 44% by ¹H NMR;

(3) β-elimination depolymerization of HsFG: To the reaction solutionobtained in the step (2), a freshly prepared 16.0 mL of 0.08 M sodiumethoxide-ethanol solution was added, and stirred at room temperature for30 min.

(4) Sodium salt conversion and carboxylic ester hydrolysis of thedepolymerized product: 67 mL of saturated sodium chloride solution and536 mL of absolute ethanol were added to the reaction solution obtainedin the step (3), centrifuged at 4000 rpm×10 min; the obtainedprecipitate was dissolved in water (125 mL), 1.05 mL of 6 M NaOHsolution was added, stirred at room temperature for 30 min, and thenneutralized by dropwise addition of 6 M HCl (pH ˜7.0). The reactionsolution was filtered through a 0.45 m filter, and the filtrate wasultrafiltered through a 30 kDa ultrafiltration membrane package. Theultrafiltrate was desalted by G25 gel column chromatography andlyophilized to obtain 1.623 g of depolymerized product dHsFG′ (yield46.4%).

(5) Isolation and purification of Compounds B6˜B8: 1 g of depolymerizedproduct dHsFG′ was dissolved in 10 mL of 0.2 M NaCl, loaded on a Bio-GelP-10 gel column (Ø2 cm, l 200 cm), and eluted with 0.2 M NaCl solutionat a flow rate of 15 mL/h. The eluate fractions of 2.5 mL/tube werecollected. Ultraviolet spectrophotometry (max 234 nm) was used formonitoring and the same eluate fractions were combined. HPGPC (TSK G2000SW column) was used to detect the purity and composition ofchromatographic samples. The unpurified samples were further purified byBio-Gel P-10 column chromatography. The purified oligosaccharides weredesalted on a Sephadex G-10 or Bio-Gel P-2 gel column and thenlyophilized.

(6) Spectral analysis: By the same method described in 1.2 (6) ofExample 1, ¹H-/¹³C- and 2D-NMR were detected using Bruker DRX 800 MHzNMR spectrometer, Q-TOF MS was analyzed using microTOF-QII ESI-MS(Bruker, Germany) mass spectrometer. The detected data were analyzedusing Bruker Compass Data-Analysis 4.0 (Bruker-Daltonics, Germany)software.

3.3 Results

(1) 47 mg of Compound B6, 55 mg of B7, 35 mg of B8 were obtainedaccording to the treatment procedure described in 3.2. The purity wasdetected to be >99% by HPGPC method (area normalization method).

(2) Structural analysis of Compounds B6˜B8: The HPGPC profiles ofCompounds B6 B8 are shown in FIG. 10. Combined with ¹H-/¹³C-/2D-NMR andQ-TOF MS analysis, Compounds B6, B7 and B8 areL-Fuc_(3S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(3S4S)-(α1,3)]-D-GlcA-(β1,4)}_(n)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(3S4S)-(α1,3)]-D-GlcA(wherein n=0, 1 and 2, namely pentasaccharide, octasaccharide andhendecasaccharide), having the chemical structural formula of:

In B6, n=0; in B7, n=1; in B8, n=2.

[Example 4] Preparation of Compounds A6, A7 and A8

L-Fuc_(2S4S)-(α1,3)-[6-Me-ΔUA-(α1,3)]-{D-Gal-NAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)-]-D-6-Me-GlcA-(β1,4)}₂-D-GalNAc_(4S6S)-ol,andL-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNS_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)-]-D-GlcA-(β1,3)}₂-D-GalNS_(4S6S)-olandL-Fuc_(2S4S)-(α1,3)-[6-Me-ΔUA-(α1,3)]-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)-]-D-6-Methyl-GlcA-(β1,3)}₂-D-1-Me-GalNAc_(4S6S)-ol

4.1 Materials

Compound A2, its preparation method and chemical structure were the sameas described in Example 1.

Hydrazine sulfate, hydrazine hydrate, Et₃N.SO₃ ([Et₃N—SO₃H]Cl,N,N-diethyl-N-sulfoethanammonium chloride) were all commerciallyavailable analytical reagents.

4.2 Methods and Results

(1) Preparation of Compound A6: 10 mg of Compound A2 was dissolved in0.5 mL of water, converted into H+ type by a Dowex 50X8 hydrogen-typecation exchange resin column, and the eluate was neutralized withtetrabutylammonium hydroxide and lyophilized to obtain 19 mg of A2tetrabutylammonium salt. The obtained A2 tetrabutylammonium salt wasdissolved in 1 mL of dimethyl sulfoxide (DMSO), 15 μL of 2 Mtrimethylsilyldiazomethane (TMSD) was added and reacted for 60 min atroom temperature, 15 μL of acetic acid was added to remove the remainingTMSD, 4 mL of absolute ethanol was added at 4° C., and centrifuged at4000 rpm×30 min, and the obtained precipitate was dissolved in 1 mL ofwater, and converted into sodium-type by a Dowex/r50w×8 50-100 (Na+type) exchange resin. The obtained product was desalted on a SephadexG-10 column and lyophilized to obtain 8.35 mg of A6. ¹H-/¹³C- and 2D-NMRwere detected by the method described in 1.2 (6) of Example 1, and thestructure of Compound A6 (the methyl ester group signal on ΔUA waslocated at 3.70 ppm) was confirmed to beL-Fuc_(2S4S)-(α1,3)-[6-Methyl-ΔUA-(α1,3)]-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)-]-D-6-Methyl-GlcA-(β1,4)}₂-D-GalNAc_(4S6S)-ol,having the structural formula of:

(2) Preparation of compound A7: 10 mg of Compound A2 was added with 2.5mg of hydrazine sulfate and 0.25 mL of hydrazine hydrate, and stirredunder nitrogen atmosphere at 105° C. for 15 h; 0.5 mL of 16% NaClsolution and 3 mL of absolute ethanol were added to the reactionsolution, and centrifuged at 4000 rpm×20 min. The resulting precipitatewas dissolved in 10 mL of water and dialyzed with a 500-1000 Da dialysisbag. The retentate was lyophilized to obtain 8 mg of deacetylatedproduct. The NMR spectrum was detected by the method described in 1.2(6) of Example 1, and the chemical structure of the deacetylated product(the Ac methyl signal at 2.0 ppm disappeared and the H signal at the2-position of GalNH₂ appeared at 3.0 ppm) was confirmed to be:

The A2 deacetylated product was dissolved in 1 mL of water, 36 mg ofNa₂CO₃ was added and heated to 55° C., and 15 mg of Et₃N.SO₃ was addedat 0, 5 and 10 h after the start of the reaction, respectively. Thereaction mixture was stirred at 55° C. for 15 h. Then 1 mL of 16% NaClsolution and 8 mL of absolute ethanol were added to the reactionsolution, and centrifuged at 4000 rpm×20 min; the precipitate wascollected and dissolved in 10 mL of water, and then dialyzed with a500-1000 Da dialysis bag. The dialysis retentate was lyophilized toobtain 6.8 mg of N-sulfated product A7.

¹H-/¹³C- and 2D-NMR were detected according to method as described in1.2(6) of Example 1, and the chemical structure of Compound A7 wasconfirmed to beL-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNS_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)-]-D-GlcA-(β1,3)}₂-D-Gal-NS_(4S6S)-ol.The structural formula is:

(3) Preparation of Compound A8: 20 mg of Compound A2 was added withDowex 50X8 hydrogen type cation exchange resin as a catalyst, and then 5mL of methanol solution was added, and heated to reflux under a nitrogenatmosphere overnight. The resin was removed by filtration, and thefiltrate was evaporated to remove the solvent to obtainC1-hydroxyalkylated product of A2. The product was converted into H+type by a Dowex 50X8 cation exchange resin column, and the eluate wasneutralized with tetrabutylammonium hydroxide and lyophilized to obtain37 mg of tetrabutylammonium salt of the C1 hydroxyalkylated product ofA2. The obtained product was dissolved in 2 mL of DMSO, added with 30 μLof 2 M TMSD, and reacted for 60 min at room temperature. Then 30 μL ofacetic acid was added to remove the remaining TMSD, and 2 mL of 16% NaClsolution and 8 mL of absolute ethanol were added at 4° C., centrifugedat rpm×30 min, the obtained precipitate was dissolved in 2 mL of waterand converted into sodium type by a Dowex/r50w×8 50-100 (Na+ type)exchange resin column. The obtained product was desalted on a SephadexG-10 column and lyophilized to obtain 13.5 mg of A8.

¹H-/¹³C- and 2D-NMR were detected by the method as described in 1.2 (6)of Example 1, and the structure of compound A8 (the methyl signal of ΔUAmethyl ester was at 3.70 ppm, and the methyl signal at the reducingterminal was at 3.23 ppm) was confirmed to beL-Fuc_(2S4S)-(α1,3)-[6-Methyl-ΔUA-(α1,3)]-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)-]-D-6-Methyl-GlcA-(β1,3)}₂-D-1-Methyl-Gal-NAc_(4S6S)-ol,having the structural formula of:

Similarly, according to the method of the present example, an alcoholcorresponding to a C2-C6 linear or branched alkane or an alkene may beselected to prepare the corresponding hydroxyalkylated product A8′.

[Example 5] Preparation of Compounds B9, B10 and B11

L-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-D-Gal-NAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-GlcA-(β1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-β1-Bn-GlcA,L-Fuc_(2S4S)-(α1,3)-L-6-Me-ΔUA-(α1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-6-Me-ΔUA-GlcA-(β1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-β1-Bnz-6-Me-GlcAandL-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-GlcA-(β1,3)}₂-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-1-deoxy-1-amino-GlcA-ol-N-4-benzoicethyl ester

5.1 Materials

Using SvFG as a starting material, hendecasaccharide (B3′) was preparedaccording to the method described in Example 2, and octasaccharide (B7′)was prepared according to the method described in Example 3.

Ethyl 4-aminobenzoate, tetrabutylammonium hydroxide, dichloromethane,pyridine, acetic anhydride, benzyl alcohol, boron trifluoride, ether,and so on were all commercially available analytical reagents.

5.2 Methods and Results

(1) Preparation of Compound B9: 40 mg of Compound B7′ was dissolved in 4mL of water, converted into H⁺ type by a Dowex 50X8 hydrogen type cationexchange resin column, and the eluate was neutralized withtetrabutylammonium hydroxide and lyophilized to obtain 80 mg of B7′tetrabutylammonium salt. The obtained B7′ tetrabutylammonium salt wasadded with 8 mL of pyridine and 8 mL of acetic anhydride, stirred at100° C. for 30 min, and blowing-dried with nitrogen at room temperature.The residue was dissolved in 4 mL of dichloromethane, added with 128 μLof benzyl alcohol, and then added dropwise with 20 μL of borontrifluoride etherate (BF₃OEt₂) at 0° C., and heated under reflux for 36h. The reaction was terminated by adding water. After shaking andstanding, the CH₂Cl₂ layer was taken and evaporated to dryness to removeCH₂Cl₂. The residue was added with 4 mL of 0.02 M sodiummethoxide-methanol solution at room temperature, stirred for 10 min toremove acetyl; evaporated to dryness to remove methanol, converted intoH+ type by a Dowex 50X8 hydrogen-type cation exchange resin column. Theeluate was neutralized with sodium hydroxide, and isolated and purifiedby Bio-gel P6, and the sugar-containing samples were combined,concentrated and desalted on a Sephadex G-10 column and lyophilized toobtain 24 mg of Compound B9.

¹H-/¹³C- and 2D-NMR were detected by the same method as described in 1.2(6) of Example 1, and the structure of Compound B9 (the benzyl-CH₂signal was at 4.6 ppm and the benzene ring signal was at 7.3 ppm) wasconfirmed to beL-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-GlcA-(j1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-1-Benzyl-GlcA-ol,having the structural formula of:

(2) Preparation of Compound B10: 10 mg of Compound B9 was dissolved in0.5 mL of water, converted into H+ type by a Dowex 50X 8 hydrogen typecation exchange resin column, and the eluate was neutralized withtetrabutylammonium hydroxide and lyophilized to obtain 19 mg of B9tetrabutylammonium salt. The obtained B9 tetrabutylammonium salt wasdissolved in 1 mL of dimethyl sulfoxide (DMSO), added with 15 μL of 2 Mtrimethylsilyldiazomethane (TMSD), and reacted at room temperature for60 min, then added with 15 μL of acetic acid to remove the remainingTMSD. 4 mL of absolute ethanol was added at 4° C., centrifuged at 4000rpm×30 min, and the obtained precipitate was dissolved in 1 mL of waterand converted into sodium type by Dowex/r50w×8 50-100 (Na+ type)exchange resin. The obtained product was purified with Bio-Gel P-6,desalted on a Sephadex G-10 column and lyophilized to obtain 8.35 mg ofB10.

¹H-/¹³C- and 2D-NMR were detected by the method as described in 1.2 (6)of Example 1, and the structure of compound B10 (methyl signal ofcarboxyl ester was at 3.7 ppm, —CH₂ signal of benzyl was at 4.6 ppm andthe benzene ring signal was at 7.3 ppm) was confirmed to beL-Fuc_(2S4S)-(α1,3)-L-6-Methyl-ΔUA-(α1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-6-Methyl-ΔUA-GlcA-(β1,3)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-β-1-Benzyl-6-Methyl-GlcA,having the structural formula of:

(3) Preparation of compound B11 330 mg of ethyl 4-aminobenzoate wasdissolved in 80 μL of a mixture solution of glacial acetic acid andmethanol (1:9), and 70 mg of sodium cyanoborohydride was added anddissolved. 20 mg of Compound B3′ was dissolved in 2 mL of water, reactedwith the mixed solution at 60° C. for 4 h in a constant temperaturewater bath, extracted with 2 mL of chloroform, and the aqueous phase waspurified by Bio-gel P10 column chromatography, desalted by a SephadexG-10 column and lyophilized to obtain about 14 mg of compound B11.

¹H-/¹³C- and 2D-NMR were detected by the method as described in 1.2 (6)of Example 1, and the structure of compound B11 (the —CH₃ and —CH₂signals of carbethoxy were located at 1.3 ppm and 4.3 ppm, respectively,and the benzene ring signals were divided into two groups at 6.78 ppmand 7.68 ppm, respectively) was confirmed to beL-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-GlcA-(β1,3)}₂-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-1-deoxy-1-amino-GlcA-ol-N-4-benzoicethyl ester, having the structural formula of:

Similarly, when benzyl alcohol is replaced by a corresponding C8-C12aromatic alcohol (for example, p-methylbenzyl alcohol, p-pentylbenzylalcohol), a series of derivatives B9′ having the corresponding C8-C12aromatic hydrocarbon group were obtained according to the preparationmethod of B9 in this Example; when ethyl 4-aminobenzoate is replaced by4-amino-aromatic (C8-C12) carboxylate (propyl 4-aminobenzoate, pentyl4-aminobenzoate), a series of derivatives B11′ having the correspondingC8-C12 aromatic hydrocarbon group were obtained according to thepreparation method of B11 in this Example.

[Example 6] Preparation of Oligosaccharide Mixture C1

6.1 Materials

SvFG, obtained as described in Example 1.

The reagents used such as benzethonium chloride, benzyl chloride, DMF,sodium hydroxide, sodium borohydride, sodium chloride, and ethanol areall commercially available analytical reagents. Sephadex G-50 (medium,50-100 m), GE Healthcare product.

6.2 Methods

(1) Quaternary ammonium salt conversion of SvFG: 70 g of SvFG wasdissolved in 1 L of water; and 175 g of benzethonium chloride wasdissolved in 2.8 L of water. The SvFG solution was titrated with abenzethonium chloride solution with stirring. After the completion ofthe titration, the mixture was centrifuged, and the precipitate waswashed three times with deionized water and dried under vacuum to obtain210 g of SvFG quaternary ammonium salt.

(2) Carboxyl esterification of SvFG: The SvFG quaternary ammonium saltobtained in the step (1) was dissolved in 1.020 L of DMF, added with 29mL of benzyl chloride, and stirred at 35° C. for 24 h, and then thereaction solution was allowed to stand and cool down to room temperature(25° C.). Sample was taken for detecting ¹H NMR spectrum and the degreeof carboxyl esterification of FG was calculated to be about 46%;

(3) β-elimination depolymerization of SvFG in the presence of a reducingagent: To the reaction solution of the step (2), a freshly prepared 333mL of 0.08 M sodium ethoxide-ethanol solution containing 0.4 M NaBH₄ wasadded, and stirred at room temperature for 30 min.

(4) Post-treatment: To the reaction solution obtained in the step (3),1.333 L of a saturated sodium chloride solution and 10.7 L of absoluteethanol were added, and centrifuged at 4000 rpm×10 min, and the obtainedprecipitate was dissolved in 5 L of water, added with 40 mL of 6 M NaOHsolution, and reacted for 30 min at room temperature. Then 6 M HCl wasdropwise added to neutralize the reaction solution (pH ˜7.0). Theobtained product was ultrafiltered through a 0.1 m² 10 kDa and 3 kDaultrafiltration membrane pack (Millipore) to remove macromolecular andsmall molecular impurities, to obtain 35 g of oligosaccharide mixtureC1.

(5) Spectral analysis: ¹H-/¹³C-/2D NMR spectra were detected accordingto the method described in 1.2 (6) of Example 1.

6.3 Results

(1) 35 g of oligosaccharide mixture C1 was obtained according to thedescribed method, with a yield of 50%;

(2) HPGPC analysis showed that C1 contained hexasaccharide,nonasaccharide, dodecasaccharide, pentadecasaccharide,octadecasaccharide and heneicosasaccharide, which were 14.2%, 23.1%,4.1%, 16.0%, 8.9%, and 5.1%, respectively.

(3) The ¹³C NMR spectrum and assignments for the oligosaccharide mixtureC1 is shown in FIG. 11. In the ¹H NMR of the oligosaccharide mixture C1,three strong signal peaks were observed in the range of 5.4˜5.7 ppm,wherein the signal peak at 5.77 ppm was H-4 position signal at thenon-reducing terminal ΔUA of the oligosaccharide mixture C1. The signalsat 5.6 ppm and 5.43 ppm were a terminal hydrogen signal of L-Fuc_(2S4S)in the sugar chain near the reducing terminal and a terminal hydrogensignal of L-Fuc_(2S4S) attached to the non-reducing terminal ΔUA,respectively.

By the signal analysis of the reducing terminal, in particular thecarbon signal analysis of the C1 position (—CH₂) of -D-GalNAc_(4S6S)-oland -D-GlcA-ol, the content of the oligosaccharide compound having-D-GalNAc_(4S6S)-ol at the reducing terminal structure was greater than95%.

In combination with the ¹³C-NMR and 2D-NMR analysis, C1 is composed ofhomologous oligosaccharide compounds, having the structure ofL-Fuc_(2S4S)-(α1,3)-L-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(2S4S)-(α1,3)]-D-GlcA-(1,3)}_(n)-D-GalNAc_(4S6S)-ol(n is a natural number), wherein the total content of the compounds ofn=1˜7 is about 95%.

[Example 7] Preparation of Oligosaccharide Mixtures D1 and D2

7.1 Materials

HsFQG, derived as described in Example 2.

The reagents used such as benzethonium chloride, benzyl chloride, DMF,DMSO, TMSD, sodium hydroxide, sodium borohydride, sodium chloride, andethanol were all commercially available analytical reagents.Ultrafiltration membrane (0.5 m²) with molecular weight cutoff of 30kDa, 10 kDa, 3 kDa, Merk Millipore.

7.2 Methods

(1) HsFG quaternary ammonium salt conversion: 330 g of HsFG wasdissolved in 4.9 L of water; 825 g of benzethonium chloride wasdissolved in another 13.2 L of water, the resulting solution was addedto the HsFG solution under stirring, centrifuged at 4000 rpm for 10 min.The precipitate was washed three times with 9 L of deionized water andvacuum dried to obtain 804 g of HsFG quaternary ammonium salt.

(2) Carboxyl esterification of HsFG: The HsFG quaternary ammonium saltobtained in the step (1) was placed in a 30 L reactor, dissolved in 3.9L of DMF. 97 mL of benzyl chloride was added at 35° C., stirred for 24h, and then the reaction solution was allowed to stand and cool down toroom temperature (25° C.). Sample was taken for ¹H NMR detection and thedegree of carboxyl esterification was calculated to be about 46%;

(3) β-elimination depolymerization and terminal peeling reaction ofHsFG: a freshly prepared 1.3 L of 0.08 M sodium ethoxide-ethanolsolution was added to the reaction solution of step (2), stirred at roomtemperature for 30 min, and then 2.5 mL of 2 M NaOH solution was addedto the reaction solution and stirred at 60° C. for 90 min.

(4) Post-treatment: 5.36 L of saturated sodium chloride solution and 57L of absolute ethanol were added to the reaction solution of the step(3), centrifuged at 4000 rpm×10 min. The resulting precipitate wasdissolved in 14.5 L of water, added with 122 mL of 6 M NaOH, stirred atroom temperature for 30 min, and then added with 54.3 g of NaBH₄,stirred at room temperature for another 30 min; and dropwise added with6 M HCl to neutralize the reaction solution (pH ˜7.0). The obtainedreaction solution was filtered through a 0.45 μm membrance filter, andthe filtrate was sequentially ultrafiltered with 0.5 m² of 30 kDa (toobtain filtrate), 10 kDa (to obtain filtrate), and 3 kDa (to obtainretentate) ultrafiltration membrane package (Millipore product) andlyophilized, thereby obtaining an oligosaccharide mixture D1 (98.7 g).[Note: After the detection, the undepolymerized macromolecularcompositions contained in the retentate obtained from ultrafiltrationthrough a 30 kDa ultrafiltration membrane contains fucan andhexosamine-containing polysaccharides].

(5) D1 carboxymethylation: 20 g of oligosaccharide mixture D1 wasdissolved in 300 mL of water, 800 mL of 6.25% benzethonium chloridesolution was added with stirring, allowed to stand and then centrifugedat 4000 rpm×10 min. The precipitate was washed three times with 300 mLof deionized water and vacuum dried to obtain 58 g of D1 quaternaryammonium salt. The obtained D1 quaternary ammonium salt was dissolved in5.8 L of DMSO, added with 87 mL of 2 M TMSD, stirred for 60 min at roomtemperature, and then added with 87 mL of acetic acid to remove theremaining TMSD; 5.9 L of saturated sodium chloride solution and 63 L of95% ethanol was sequentially added under stirring, centrifuged at 4000rpm×30 min. The resulting precipitate was dissolved in 2 L of deionizedwater, desalted by ultrafiltration through a 3 kDa ultrafiltrationmembrane, and the retentate was lyophilized to obtain D2 (16.3 g).

(6) Spectral analysis: ¹H-/¹³C- and 2D-NMR were detected according tothe method described in 1.2 (6) of Example 1.

7.3 Results

(1) Yield and Chemical Composition Analysis of Oligosaccharide MixtureD1

98.7 g of oligosaccharide mixture D1 was obtained according to themethod, with a yield of about 30%.

HPGPC analysis (FIG. 12) showed that D1 contained pentasaccharide,octasaccharide, hendecasaccharide, tetradecasaccharide,heptadecasaccharide, eicosasaccharide, which were 4.3%, 17.1%, 18.0%,16.3%, 14.1%, and 11.1%, respectively. The total content ofpentasaccharide˜nonacosasaccharide was about 96%.

The ¹³C-NMR spectrum of the oligosaccharide mixture D1 is shown in FIG.13. In the ¹H-NMR of D1, there was a strong signal at 5.685 ppm, whichwas from 4-position hydrogen of ΔUA. There were three strong signalpeaks at 5.0˜5.6 ppm (5.283, 5.201 and 5.030 ppm), which were thea-anomeric proton signals of L-Fuc_(3S4S) linked to D-GlcA, ΔUA andD-GlcA-ol, respectively. By analyzing the terminal hydrocarbon signal ofthe reducing terminal glycosyl group, the content of the oligosaccharidecompound having -D-GlcA-ol at the reducing terminal glycosyl group in D1was more than 95%.

Combined with ¹³C- and 2D-NMR analysis, it can be seen that the mixtureD1 was a mixture of homologous oligosaccharide compounds having astructure ofL-Fuc_(3S4S)-(α1,3)-L-ΔU-(α1,3)-{D-GalNAc_(4S6S)-(β3,4)-[L-Fuc_(3S4S)-(α1,3)]-D-GlcA-(β1,3)}_(n)-D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(3S4S)-(a1,3)]-D-GlcA-ol (n is a natural number).

(2) Analysis of Yield and Chemical Composition of OligosaccharideMixture D2

16.3 g of oligosaccharide mixture D2 was obtained according to themethod, with a yield of about 80%;

HPGPC analysis showed that it contained pentasaccharide, octasaccharide,hendecasaccharide, tetradecasaccharide, heptadecasaccharide,eicosasaccharide, which were 3.34%, 16.71%, 17.03%, 17.25%, 13.78%, and12.25%, respectively.

Compared to the ¹H NMR spectrum of D1, new methyl (ester) group signalslinked to hexuronic acid (-ol) appeared in the ¹H NMR of D2, which wereat 3.7 ppm and 3.2 ppm, respectively. In combination with ¹H-/¹³C- and2D-NMR analysis, it can be seen that the mixture D2 is a mixture ofhomologous oligosaccharide compounds having a structure ofL-Fuc_(3S4S)-(α1,3)-L-6-Me-ΔUA-(α1,3)-{D-GalNAc_(4S6S)-(β1,4)-[L-Fuc_(3S4S)-(α1,3)]-D-6-Me-GlcA-(β1,4)}_(n)-D-GalNAc_(4S6S)-(α1,3)-[L-Fuc_(3S4S)-(α1,3)]-D-6-Me-GlcA-ol(n is a natural number). Wherein, the sum of the contents of thecompounds of n=1˜9 is about 96%.

[Example 8] Analysis of Anticoagulation and Coagulation FactorInhibitory Activity

8.1 Materials

Samples: oligosaccharide compounds A1˜A8, B1˜B11, oligosaccharidemixtures C1, D1, and D2, prepared according to the method described inExamples 1˜7.

Control: Enoxaparin Sodium Injection (LMWH, Mw 3500˜5500 Da,Sanofi-Aventis product);

Reagents: coagulation-controlled plasma (047B-D024A), activated partialthromboplastin time (APTT), prothrombin time (PT) assay kits, all ofwhich were TECO GmbH company (Germany) products; Factor VIII test kit,Heparin Cofactor II (HCII), AT-dependent anti-factor IIa detection kit,AT-dependent anti-factor Xa detection kit, thrombin (factor IIa),thrombin substrate CS01 (38), KK substrate CS31 (02) were HYPHEN BioMedcompany (France) products; Factor VIII (FVIII), Bayer Healthcare LLC(Germany) product; ADP, Chronolog company (USA) product; sodium citrate,chloral hydrate, natural saline, were all commercial reagents.

Instruments: XS105 electronic balance, FE20 pH meter, METTLER TOLEDOproducts; HH-4 constant temperature water bath, Gongyi Yuhua companyproduct, China; VOR76X-6 vortex oscillator, Hainan Qilin Bell product;Spectrafuge-24 D907386 centrifuge, Labnet product; MC-4000 bloodcoagulation instrument, TICO GmbH company (Germany) product; MicroplateReder ELx 808 microplate reader, Bio-Tek company product; Chronolog-700platelet aggregation instrument, Chrono-log company (USA) product.

8.2 Methods

(1) Preparation of sample solution: Oligosaccharide compounds A1˜A8,B1˜B11 and oligosaccharide mixtures C1, D1, D2 were all dissolved inTris-HCl buffer and diluted to the desired series of solubility.

(2) Anticoagulant activity assay: 90 μL of human-controlled plasma wasadded to the sample or 10 μL of the control solution, and then theclotting time (APTT and PT) was detected by the MC-4000 coagulometeraccording to the method described in the APTT and PT kit instructions.

(3) Coagulation Factor Inhibitory Activity Analysis:

Xase inhibitory activity assay: Detection was performed according to kitinstructions and literature methods by combining the Factor VIII andFactor VIII detection kits. Specifically, to each well of a 96-wellplate, 30 μL of test solution, control solution, or Tris-HCl buffer(negative control) was added, and 30 μl of FVIII (2 IU/ml), 30 μl of R₂(60 nM FIXa, containing FIIa, PC/PS, Ca²⁺) were sequentially added,mixed by shaking the plate, incubated at 37° C. for 2 min; and then 30μL of R₁ (50 nM FX, containing direct thrombin inhibitor) was added,mixed by shaking the plate, incubated at 37° C. for 1 min; and then 30μL of R₃ (FXa chromogenic substrate SXa-11, about 8.4 mM) was added. Theabsorbance at 405 nm (OD₄₀₅) was detected with a microplate reader,continuously measuring for 7.5 min at a interval of 30 s. The Xaseactivity and IC₅₀ value of Xase inhibition of the test sample werecalculated based on the OD₄₀₅ change value.

AT-dependent Xa inhibitory activity assay: Heparin Anti-FIIa kit wasused for detection. To a 96-well plate, 30 μL of sample, controlsolution or Tris-HCl buffer (negative control) was added, then 30 μL of1 IU/mL AT solution was added, mixed well and incubated at 37° C. for 1min; and than 30 μL of 8 μg/mL FXa solution was added, mixed well andincubated at 37° C. for 1 min, then 30 μL of pre-warmed 1.25 mM Xachromogenic substrate SXa-11 was added. OD₄₀₅ was detected by amicroplate reader.

AT-dependent IIa inhibitory activity assay: Heparin Anti-FIIa kit wasused for detection. To a 96-well plate, 30 μL of sample, controlsolution or Tris-HCl buffer (negative control) was added, and then 30 μLof 1 IU/mL AT solution was added, mixed well by shaking the plate andincubated at 37° C. for 2 min; 30 μL of 24 IU/mL FIIa solution wasadded, mixed well by shaking the plate and incubated for 2 min at 37°C., and then 30 μL of pre-warmed 1.25 mM FIIa specific chromogenicsubstrate CS-01 (38) was added, mixed well by shaking the plate. TheOD₄₀₅ was detected by a microplate reader and the IC₅₀ value of FIIainhibition of each sample was calculated.

HC-II-dependent IIa inhibitory activity assay: 30 μL of sample, controlsolution or Tris-HCl buffer (negative control) was added, 30 μL of 1 μMHCII solution was added, and incubated at 37° C. for 2 min; and then 30μL of 20 NIH/mL FIIa was added, and incubated at 37° C. for 1 min; andfinally 30 μL of pre-warmed 4.5 mM FIIa chromogenic substrate CS-01 (38)was added. OD₄₀₅ was detected by a microplate reader and the IC₅₀ valueof FIIa inhibition of each sample was calculated.

Data processing: The average value of OD₄₀₅ detected by the duplicatedwell was used as the detection value of the test sample and thereference of each concentration, and the slope of the linear fit betweenthe detected value to the time value (the change rate of the absorbancevalue OD₄₀₅/min) indicated enzymatic activity of coagulation factor.Taking the clotting factor activity of the negative control well as100%, coagulation factor activity (percentage) in the presence of thetest sample was calculated. The coagulation factor activity in thepresence of the test sample was plotted against the concentration of thetest sample, and fitted according to the following formula, to calculatethe IC₅₀ value:

B=(IC₅₀)^(n)/{(IC₅₀)^(n)+[I]^(n)}

in the formula, B is the coagulation factor activity (percentage) in thepresence of the test sample, [I] is the concentration of the testsample, IC₅₀ is the half inhibitory concentration (concentration of thetest sample required to inhibit 50% of the activity), and n is the Hillcoefficient.

(4) Effect on Surface Activation and Platelet Activity:

FXII activation activity assay: To a 96-well plate was added 30 μL ofseries concentration sample and reference solution, respectively, andthen 30 μL of human standard plasma that was diluted 4 times with a 0.02M Tris-HCl (pH 7.4) buffer containing 0.15 M NaCl was added, andincubated at 37° C. for 2 min, and then 30 μL of 6 mM kallikreinchromogenic substrate CS-31 (02) was added, and the OD₄₀₅ value wasdetected by a microplate reader.

Platelet activation activity test: Anticoagulated blood was collectedfrom healthy volunteers to prepare platelet-rich plasma (PRP) andplatelet-poor plasma (PPP). Chronolog-700 platelet aggregationinstrument and turbidimetry were used to detect platelet-inducedaggregation activity of serial concentration solutions of the testsample, which were prepared by dissolving in natural saline.

8.3 Results

Anticoagulation and coagulation factor inhibitory activity: The resultsare shown in Table 5. The oligosaccharide compounds and the mixturethereof according to the present invention have significant prolongedAPTT activity, without affecting PT and TT, indicating that they canhave significant anticoagulant activity against intrinsic coagulationpathway, and have no significant effect on extrinsic coagulation. Theoligosaccharide compounds and the mixture thereof according to thepresent invention have significant inhibitory activity on factor Xase;in the presence or absence of antithrombin (AT), they have nosignificant effect on coagulation factors such as coagulation factorsIIa, Xa, XIIa, but may have a certain intensity of heparin cofactor II(HC-II)-dependent IIa inhibitory activity.

An alcohol corresponding to a C2-C6 linear or branched alkane or alkenewas selected to prepare the corresponding hydroxyalkylated product A8′according to Example 4. Studies on the activity of these series ofderivatives show that they have similar activity to A8, that is, theyhave prolonged APTT activity (with the drug concentration for doublingthe APTT clotting time being 7.0-10 μg/mL), without affecting PT and TT;have significant selective inhibitory activity against factor Xase(IC₅₀, 50-100 ng/mL), have no significant effect on coagulation factorssuch as factor IIa, Xa, XIIa, and have a certain intensity of heparincofactor II (HC-II)-dependent IIa inhibitory activity.

According to the preparation method of Example B9, a series ofderivatives B9′ having a corresponding C8-C12 aromatic hydrocarbon groupwere obtained, and according to the preparation method of Example B11, aseries of derivatives B11′ having a corresponding C8-C12 aromatichydrocarbon group were obtained. They have similar activities to B9 andB11, respectively; have drug concentration of doubling APTT clottingtime of 6.0-9 μg/mL, without affecting PT and TT; have significantselective inhibitory activity against factor Xase (IC₅₀, 40-110 ng/mL),and have a certain intensity of heparin cofactor II (HC-II)-dependentIIa inhibitory activity.

TABLE 5 Anticoagulant and coagulation factor inhibitory activity ofoligosaccharide compounds and oligosaccharide mixtures Drugconcentration required for multiplication of Drug concentration requiredcoagulation time to inhibit 50% of coagulation (μg/mL) factor activity(IC₅₀, ng/mL) APTT PT TT Xase Xa (AT) Ha (AT) Ha (HC-II) A1 60.1 # # #### ## 450 A2 7.2 # # 60.5 ## ## 323 A3 6.8 # # 39.8 ## ## 258 A4 5.3 # #28.2 ## ## 231 A5 3.9 # # 23.6 ## ## 320 A6 7.5 # # 68.4 ## ## 385 A76.9 # # 58.9 ## ## 298 A8 8.2 # # 70.6 ## ## 335 B1 53.9 # # 850 ## ##705 B2 7.5 # # 64.2 ## ## 753 B3 6.1 # # 31.1 ## ## 402 B4 4.5 # # 20.6## ## 408 B5 4.0 # # 19.8 ## ## 365 B6 50.2 # # 1670 ## ## 817 B7 7.3 ## 58.3 ## ## 432 B8 5.8 # # 29.6 ## ## 412 B9 7.6 # # 59.6 ## ## 706 B107.9 # # 61.5 ## ## 721 B11 6.3 # # 30.3 ## ## 375 C1 4.0 # # 22.3 ## ##404 D1 4.3 # # 21.2 ## ## 230 D2 5.1 # # 23.6 ## ## 278 LMWH 7.8 64 4.0120.0 16 36 431 Note: #, >128 μg/mL; ##, >5000 ng/mL

(2) Effect on Surface Activation and Platelet Activity:

XII activation activity analysis: Within the concentration range of notmore than 100 μg/ml, all the oligosaccharide compounds andoligosaccharide mixtures have no significant XII activation activity;

Platelet activation activity assay: Within the concentration range ofnot more than 50 μg/ml, all oligosaccharide compounds andoligosaccharide mixtures have no significant platelet activationactivity.

[Example 9] Effect on Antithrombotic Activity and Bleeding

9.1 Materials

The preparation of A2 was as shown in Example 1, and the preparation ofD1 was as shown in Example 7.

Control: Low molecular weight heparin (LMWH), Sanofi-Aventis (France)product, batch number 4SH69.

Reagents: chloral hydrate (hydrated trichloroacetaldehyde), SinopharmChemical Reagent Co., Ltd.; natural saline, Kunming NanjiangPharmaceutical Co., Ltd.

Experimental animals: SD rats, weighing 250˜350 g, male, provided byHunan Slack Jingda Experimental Animal Co., Ltd., license number SCXK(Xiang) 2011-0003; New Zealand rabbits provided by Kunming MedicalUniversity, SCXK (Dian) 2011-0004, used to make rabbit brain powderinfusion.

9.2 Methods

9.2.1 Anti-Venous Thrombosis Experiment

Grouping and Administration: Rats were randomly divided into 8 groupswith 8 animals in each group. The experimental groups and the dose ofthe animals in each group were (1) natural saline (NS) control group;(2) LMWH 4.0 mg/kg group; (3) A2 2.5 mg/kg group; (4) A2 group 5.0mg/kg; (5) A2 10 mg/kg group; (6) D1 2.5 mg/kg group; (7) D1 5.0 mg/kggroup; (8) D1 10 mg/kg group. The rats in each group were administeredsubcutaneously (sc.) into the back, and the administration volume was 1mL/kg. The modeling experiment was performed 1 hour afteradministration.

Preparation of Rabbit Brain Powder Infusion:

A New Zealand rabbit was sacrificed, and the rabbit brain was taken outimmediately. Rabbit brain powder infusion was prepared according to theliterature method (Thromb Haemost, 2010, 103(5): 994-1004), and storedat −20° C. for use.

Induction of Inferior Vena Cava Thrombosis by Rabbit Brain PowderInfusion:

The rats were anesthetized by intraperitoneally injecting with 10%chloral hydrate (300 mg/kg), the abdominal wall was cut longitudinallyalong the midline of the abdomen, the viscera was removed, and theinferior vena cava and its branches were isolated. A ligature was passedthrough the lower margin of the left renal vein of the inferior venacava, to ligate the inferior vena cava branches below the left renalvein. The femoral vein was injected with 2% rabbit brain powder infusion(1 mL/kg). After 20 seconds, the lower margin of the left renal vein wasligated. After the operation, the viscera was placed back into theabdominal cavity and covered with medical gauze (infiltrated withnatural saline). After 20 minutes, the blood vessel was clamped at 2 cmbelow the ligature, and the blood vessel was longitudinally dissected totake out the thrombus. The length of the thrombus was measured, and thewet weight of the thrombus was weighed and then dry weight was weighedafter drying at 50° C. for 24 h.

Data Processing and Statistics: The SPSS software was used to organizeand analyze the data, and the measurement data were expressed asmean±standard deviation (x±s). Data normality in different groups wastested using One-Sample KS test, variance homogeneity was tested usingLevene test. If the data conformed to the normal distribution, and thevariance was uniform, the significance was judged by One-Way ANOVA,otherwise, the significance was judged by Two-Independent-Samples Test.

9.2.2 Bleeding Tendency Detection

Grouping and administration: Mice were randomly divided into 10 groupswith 8 animals in each group. The experimental groups and the dose ofthe animals in each group were (1) natural saline (NS) control group;(2) LMWH 4.0 mg/kg group; (2) LMWH 20 mg/kg group; (3) LMWH 100 mg/Kggroup; (4) A2 5 mg/kg group; (5) A2 25 mg/kg group; (6) A2 125 mg/kggroup; (7) D1 5 mg/kg group; (8) D1 25 mg/Kg group; (10) D1 125 mg/kggroup. The rats in each group were administered subcutaneously (sc.)into the back, and the dose volume was 10 mL/kg.

Test Methods:

After 60 min of subcutaneous administration in each experimental group,the mice were placed in a mouse holder, and the tail tip was cut by 5 mmby tail-clipping method, and the mouse tail was immersed in 40 mL ofpurified water (37° C.) in the beaker. Timing was started from the firstdrop of blood from the cut mouse tail, and stirring was continued. At 60min, the beaker was placed for 60 min and then the absorbance of thesolution (OD540) was detected by a UV spectrophotometer.

In addition, whole blood of healthy mice was taken, and the whole bloodof different volumes of mice was added to 40 mL of purified water,stirred uniformly and allowed to stand for 60 min. The absorbance(OD₅₄₀) of the solution was detected by the same method and thevolume-absorbance curve was plotted and used as the standard curve forcalculating the amount of bleeding. The amount of bleeding in eachexperimental group was calculated by the standard curve.

Data Processing and Statistics:

The SPSS software was used to organize and analyze the data, and thedetected data was expressed as mean±standard deviation (x±s). Datanormality in different groups was tested using One-Sample KS test,variance homogeneity was tested using Levene test. If the data conformedto the normal distribution, and the variance was uniform, thesignificance was judged by One-Way ANOVA, otherwise, the significancewas judged by Two-Independent-Samples Test.

9.3 Results

(1) Antithrombotic activity: As shown in FIG. 14, the results show thatboth A2 and D1 have significant antithrombotic activity at theexperimental dose, and the inhibition rate of thrombosis may reach above70% at the dose of 5 mg/kg˜10 mg/kg.

(2) Bleeding tendency influence: As shown in FIG. 15, under high doseadministration of equal multiple of the equivalent antithrombotic dose,the amount of bleeding in A2 and D1 administration groups issignificantly lower than that in LMWH administration group.

[Example 10] Preparation of A3 Pharmaceutical Composition as LyophilizedPowder for Injection

10.1 Materials

Compound A3, purified dodecasaccharide compound prepared according tothe method described in Example 1.

NaCl, commercially available, pharmaceutical grade; Sterile water forinjection; 2 mL medium borosilicate tube glass bottle for injection,Millipore Pellicon 2 ultrafiltration system (Merk Millipore); VirTisUltra 35 EL lyophilizer.

10.2 Formulation

Raw material (Excipient) Dosage A3 20 g NaCl  4 g H₂O 500 mL Totallyprepared into 1000 vials

10.3 Preparation Process

(1) Process procedure: Twice the prescribed amount of A3 (40 g) and NaCl(8 g) were weighed and dissolved in 1.0 L of water for injection. Afterdissolved completely under stirring, it was treated by a Milliporeultrafiltration device having an ultrafiltration membrane package with amolecular weight cut-off of 10 kDa to remove the pyrogen. In a sterileenvironment, after 0.22 μm membrane filtration and sterilization, thesolution was filled in a 2 mL vial of 0.5 mE per vial while monitoringthe filling process, partially stoppered, and placed in the drying boxof the pilot lyophilizer (VirTis, US), lyophilized according to theprogrammed lyophilization process, stoppered, withdrawn from thelyophilizer, capped, and inspected.

(2) Lyophilization process:

Pre-cooling: The samples were placed in the lyophilizer; the temperatureof shelves was dropped to −25° C., maintaining for 1 h, then dropped to−45° C., maintaining for 3 h; the temperature of cold trap was droppedto −50° C., and the vacuum degree was pumped to 40 Pa.

Sublimation: The temperature was increased uniformly to −30° C. within 1h, maintaining for 2 h; increased uniformly to −20° C. within 2 h,maintaining for 6 h; the vacuum degree was maintained at 40˜30 Pa.

Drying: The temperature was increased to −5° C. within 2 h, maintainingfor 2 h, and the vacuum was maintained at 30˜20 Pa; the temperature wasincreased to 10° C. within 0.5 h, maintaining for 3 h, and the vacuumdegree was maintained at 30˜20 Pa; the temperature was increased to 40°C. within 0.5 h, maintaining for 4 h, and the vacuum degree was pumpedto the lowest.

10.4 Results

According to the preparation process, 1,960 vials of qualified productsof A3 lyophilized preparation were obtained, and the qualified rate ofthe finished product was about 98%. After testing, the lyophilized cakehad regular appearance; the sterility, pyrogen and insoluble particulatetesting were all qualified; the moisture testing results showed that thewater content was less than about 3%, and the loading testing resultsshowed that the loading was within 95˜115% of the planned loading.

[Example 11] Preparation of D1 Pharmaceutical Composition as LyophilizedPowder for Injection

11.1 Materials

Oligosaccharide mixture D1, prepared according to the method describedin Example 7. NaCl, commercially available, pharmaceutical grade;sterile water for injection; 2 mL medium borosilicate tube glass bottlefor injection, Millipore Pellicon 2 ultrafiltration system (MerkMillipore); Lyophilizer (LYO-20 m²), Shanghai Toffion Sci &Tech Co.,Ltd.

11.2 Formulation

Raw material (Excipient) Dosage D1 50 g NaCl  9 g H₂O 1.0 L Totallyprepared into 1000 vials

11.3 Preparation Process

(1) Process procedure: 20 times the prescribed amount of D1 (1000 g) andNaCl (180 g) were weighed and dissolved in 20 L of water for injection.After dissolved completely under stirring, it was treated by a Milliporeultrafiltration device having an ultrafiltration membrane package with amolecular weight cut-off of 10 kDa to remove the pyrogen. In a sterileenvironment, after 0.22 μm membrane filtration and sterilization, thesolution was filled in a 2 mL vial of 0.5 mL per vial while monitoringthe filling process, partially stoppered, and placed in the drying boxof a production lyophilizer (VirTis, US), lyophilized according to theprogrammed lyophilization process, stoppered, withdrawn from thelyophilizer, capped, and inspected to be qualified, to obtain the finalproducts.

(2) Lyophilization Process:

Pre-cooling: The samples were placed in the lyophilizer; the temperatureof shelves was dropped to −25° C., maintaining for 1 h, then dropped to−45° C., maintaining for 3 h; the temperature of cold trap was droppedto −50° C., and the vacuum degree was pumped to 40 Pa.

Sublimation: The temperature was increased uniformly to −30° C. within 1h, maintaining for 2 h; increased uniformly to −20° C. within 2 h,maintaining for 6 h; the vacuum degree was maintained at 40˜30 Pa.

Drying: The temperature was increased to −5° C. within 2 h, maintainingfor 2 h, and the vacuum degree was maintained at 30˜20 Pa; thetemperature was increased to 10° C. within 0.5 h, maintaining for 3 h,and the vacuum degree was maintained at 30˜20 Pa; the temperature wasincreased to 40° C. within 0.5 h, maintaining for 4 h, and the vacuumdegree was pumped to the lowest.

11.4 Results:

According to the preparation process, 17,600 vials of qualified samplesof D1 lyophilized preparation were obtained, and the qualified rate ofthe finished product was about 88%.

Appearance/characteristic: This product was a white loose mass.

Loading testing: The gravimetric testing was in compliance with theregulations.

Sterility testing: An appropriate amount of this product was taken andtested according to law (1101, Volume IV, Chinese Pharmacopoeia Edition2015). The test results showed that the batch of samples met the qualityrequirements of injection.

Pyrogen testing: The product was prepared into a solution containing 3.5mg of D1 per 1 mL, and tested according to the law (1142, Volume IV,Chinese Pharmacopoeia Edition 2015), the results showed that this batchof samples met the quality requirements of pyrogen testing forinjection.

What is claimed is:
 1. An oligosaccharide compound or a pharmaceutically acceptable salt thereof, characterized in that, the oligosaccharide compound has antithrombotic activity, and has a general structure represented by Formula (I):

in the formula, R₁, R₂, R₃, R₄, and R₅ are optionally and independently —H or —SO₃H; R₆ is optionally —H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group; R₇ is optionally —H, —SO₃H, C2-C5 acyl; R₈ is optionally a group represented by Formula (II), Formula (III) or Formula (IV):

in Formula (II), Formula (III) and Formula (IV), R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are defined as above; R₉ and R₁₀ are optionally —H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group; R₁₁ is optionally —NHR₁₂, —OR₁₃, wherein R₁₂ and R₁₃ are optionally —H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group; and n is optionally 0 or a natural number of 1˜8.
 2. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when R₈ is the group represented by Formula (II), the oligosaccharide compound has a general structure represented by Formula (V):

wherein, R₁═—H, R₂═R₃═R₄═R₅═—SO₃—; or R₁═R₃═R₄═R₅═—SO₃—; R₂═—H.
 3. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when R₈ is a group represented by Formula (III), the oligosaccharide compound has a general structure represented by Formula (VI):

wherein R₁═—H, R₂═R₃═R₄═R₅═—SO₃—; or R₁═R₃═R₄═R₅═—SO₃—, R₂═—H.
 4. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, when R₈ is the group represented by Formula (IV), the oligosaccharide compound has a general structure represented by Formula (VII):

wherein: R₁═—H, R₂═R₃═R₄═R₅═—SO₃—; or R₁═R₃═R₄═R₅═—SO₃, R₂═—H.
 5. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, in the general structure represented by Formula (I), n is optionally 1, 2, 3 or
 4. 6. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, the pharmaceutically acceptable salt is optionally an alkali metal salt, an alkaline earth metal salt or an organic ammonium salt.
 7. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that, the pharmaceutically acceptable salt is optionally a sodium salt, a potassium salt or a calcium salt.
 8. An oligosaccharide mixture or a pharmaceutically acceptable salt thereof composed of the oligosaccharide compound of claim 1 and having antithrombotic activity, characterized in that, the oligosaccharide mixture is composed of homologues of the oligosaccharide compound of claim 1; and in the oligosaccharide compound of Formula (I) composing the oligosaccharide mixture, R₈ is a group represented by Formula (II), Formula (III), or Formula (IV), and in the molar ratio, the proportion of the oligosaccharide compound of Formula (I) in which R₈ is a group simultaneously represented by Formula (II), simultaneously represented by Formula (III) or simultaneously represented by Formula (IV) is not less than 95% in the mixture.
 9. A preparation method of the oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: preparing a carboxylate of natural fucosylated glycosaminoglycan, followed by subjecting the fucosylated glycosaminoglycan to “β-elimination reaction” and terminal “reduction reaction”, optionally in the presence of a strong base and a reducing agent, to obtain a mixture of the homologous oligosaccharide compounds; or subjecting the fucosylated glycosaminoglycan to “β-elimination reaction” and terminal “peeling reaction” in the presence of a strong base to obtain a mixture of the homologous oligosaccharide compounds; then isolating and purifying, and optionally performing a substituent modification to obtain the desired purified oligosaccharide compound.
 10. The preparation method according to claim 9, characterized in that: the oligosaccharide compound has the general structure represented by Formula (I) as defined in claim 1, and R₈ is the group represented by Formula (II) as defined in claim 1; the method comprises: (a) converting fucosylated glycosaminoglycan into a quaternary ammonium salt form, followed by completely or partially converting the carboxyl group on hexuronic acid residue in the obtained fucosylated glycosaminoglycan quaternary ammonium salt into a carboxylate in an organic solvent; (b) in an anhydrous organic solvent in presence of a reducing agent, treating the fucosylated glycosaminoglycan carboxylate with a strong base to cause β-elimination depolymerization and terminal reduction reaction, to obtain a mixture of homologous oligosaccharide compounds having D-acetylaminogalactitol at the reducing terminal; (c) converting the oligosaccharide mixture obtained in the step (b) into an alkali metal salt, followed by subjecting its carboxylate to basic hydrolysis in an aqueous solution, to obtain a mixture of the homologous oligosaccharide compounds containing a free carboxyl group; (d) isolating the oligosaccharide mixture obtained in the step (c) by chromatography, to obtain a purified oligosaccharide compound; (e) optionally, subjecting the purified oligosaccharide compound obtained in the step (d) to a substituent structural modification.
 11. The preparation method according to claim 10, characterized in that, in the preparation steps: in the step (a), the fucosylated glycosaminoglycan quaternary ammonium salt is benzethonium salt (N,N-dimethyl-N-[2-[2-[4(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethyl]benzylammonium salt); the organic solvent is DMF or a DMF-ethanol mixture; the carboxylate is a benzyl ester, and the complete or partial conversion into carboxylate means that the degree of carboxyl esterification of fucosylated glycosaminoglycan is in the range of from about 30% to about 100%; in the step (b), the organic solvent is DMF or a DMF-ethanol mixture; the reducing agent is sodium borohydride; the strong base is sodium ethoxide; in the step (c), the conversion of the oligosaccharide mixture to an alkali metal salt means that a saturated aqueous solution of sodium chloride is added to the reaction solution to convert the oligosaccharide mixture into a sodium salt form; the basic hydrolysis means that the carboxylate group of the homologous oligosaccharide compounds is hydrolyzed in NaOH aqueous solution with a concentration of 0.25 M˜1 M; in the step (d), the chromatography includes, but is not limited to, gel chromatography and/or ion exchange chromatography; in the step (e), the further substituent structural modification includes, but is not limited to, carboxyl esterification of D-glucuronic acid group and unsaturated hexenuronic acid group in the oligosaccharide compound; deacetylation and optional reacylation or resulfation of D-acetylgalactosamine; hydroxyalkylation at the C1 position of the reducing terminal -D-GalNAc-ol.
 12. The preparation method according to claim 11, characterized in that, the oligosaccharide compound has a general structure represented by Formula (I) as defined in claim 1, and R₈ is a group represented by Formula (III) or Formula (IV) as defined in claim 1, the method comprises the specific steps of: (a) converting fucosylated glycosaminoglycan into a quaternary ammonium salt form, followed by completely or partially converting the carboxyl group on hexuronic acid residue in the obtained fucosylated glycosaminoglycan quaternary ammonium salt into a carboxylate in an organic solvent; (b) in an anhydrous organic solvent, treating the fucosylated glycosaminoglycan carboxylate with a strong base to cause β-elimination depolymerization, followed by subjecting the depolymerized product to “peeling reaction” by adding a small amount of aqueous solution of a strong base to lose the -D-GalNAc residue at the reducing terminal, thereby obtaining a mixture of homologous oligosaccharide compounds having glycosyl group -D-GlcA at the reducing terminal; (c) converting the oligosaccharide mixture obtained in the step (b) into an alkali metal salt, and subjecting the carboxylate of the homologous oligosaccharide compounds to basic hydrolysis in an aqueous solution, to obtain a mixture of the homologous oligosaccharide compounds containing a free carboxyl group; (d) isolating and purifying the oligosaccharide compounds in the oligosaccharide mixture obtained in the step (c) by chromatography; (e) optionally, subjecting the purified oligosaccharide compound obtained in the step (d) to a further substituent structural modification.
 13. The preparation method according to claim 12, characterized in that, in the preparation steps: in the step (a), the quaternary ammonium salt is benzethonium salt; the organic solvent is DMF or a DMF-ethanol mixture; the carboxylate is benzyl ester, and “the complete or partial conversion into carboxylate” means that the degree of carboxyl esterification of fucosylated glycosaminoglycan is in the range of from about 30% to about 100%; in the step (b), the organic solvent is DMF or a DMF-ethanol mixture; the strong base is sodium ethoxide; the small amount of aqueous solution of a strong base means a 1 M˜2 M aqueous solution of NaOH that is equivalent to about ⅕ to 1/10 of the total volume of the reaction solution; in the step (c), converting the oligosaccharide mixture into an alkali metal salt means that a saturated aqueous solution of sodium chloride is added to the reaction solution to convert the oligosaccharide mixture into a sodium salt form; the basic hydrolysis means that the carboxylate of the homologous oligosaccharide compounds is hydrolyzed in NaOH aqueous solution with a concentration of 0.05 M˜1 M; in the step (d), the chromatography includes, but is not limited to, gel chromatography and/or ion exchange chromatography; in the step (e), the further substituent structural modification includes, but is not limited to, carboxyl esterification of D-GlcA and ΔUA in the oligosaccharide compound; deacetylation and optional reacylation or resulfation of D-GalNAc; alkylation, reduction, reductive amination or reductive alkylation of the hemiacetal at the C1 position of the reducing terminal -D-GlcA.
 14. A pharmaceutical composition having antithrombotic activity, characterized in that, it comprises an anti-thrombotic effective amount of an active ingredient and a pharmaceutically acceptable excipient, the active ingredient is the oligosaccharide compound or the pharmaceutically acceptable salt thereof according to claim 1, or the oligosaccharide mixture or the pharmaceutically acceptable salt thereof according to claim
 8. 15. The pharmaceutical composition according to claim 14, characterized in that, the preparation form of the pharmaceutical composition is an aqueous solution for injection or a lyophilized powder for injection, and its unit dosage form contains 20 mg˜100 mg of the active ingredient.
 16. Use of the oligosaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, or the oligosaccharide mixture or a pharmaceutically acceptable salt thereof according to claim 8 in the preparation of a medicament for the treatment and/or prevention of thrombotic diseases, the thrombotic diseases are venous thrombosis, arterial thrombosis and/or ischemic cardiovascular and cerebrovascular diseases.
 17. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claim 2, characterized in that, in the general structure represented by Formula (V), n is optionally 1, 2, 3 or
 4. 18. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claim 3, characterized in that, in the general structure represented by Formula (VI), n is optionally 1, 2, 3 or
 4. 19. The oligosaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claim 4, characterized in that, in the general structure represented by Formula (VII), n is optionally 1, 2, 3 or
 4. 